Compositions and methods for treating cancers

ABSTRACT

The present invention relates to compositions and methods for treating of cancer. In some embodiments, the invention relates to the use of agents that can modulate a component in the CDX2-KLF4 signaling pathway to treat myelodysplastic syndromes (MDS), acute myelogenous leukemia (AML), acute lymphocytic leukemia (ALL), adult T-cell leukaemia (ATLL), lymphoma, gastric cancer, multiple myeloma, or combinations thereof, or a condition associated with abnormal activity of the CDX2-KLF4 signaling pathway.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims the benefit of U.S. Provisional Application No.61/887,285, filed Oct. 4, 2013, U.S. Provisional Application No.61/919,023, filed Dec. 20, 2013, U.S. Provisional Application No.62/017,505 filed on Jun. 26, 2014, and U.S. Provisional Application No.62/037,868, filed Aug. 15, 2014, all of which are hereby incorporated byreference in their entireties.

FIELD OF THE INVENTION

The present invention relates generally to compositions and methods fortreating cancers.

BACKGROUND OF THE INVENTION

Many drugs or drug candidates have been developed for the treatment ofvarious cancers, including some small molecule compounds. However,current treatments for many cancers are not very effective in patientswith specific subsets of cancers, or are too toxic in such patients orin general.

Myelodysplastic syndromes (MDS) are a group of diseases that affect thebone marrow and blood. Some types of MDS are mild and easily managed,while other types are severe and life-threatening. Myelodysplasticsyndromes (MDS) are rarely cured; most patients never actually completetreatment. The current treatment of MDS is based on the stage and themechanism of the disease that predominates the particular phase of thedisease process. Bone marrow transplantation has been used in patientswith poor prognosis or late-stage MDS. Epstein and Slease, 1985, Surg.Ann. 17:125. This type of therapy, however, is both painful for donorand recipient, because of the involvement of invasive procedures and cancause severe and even fatal complications to the recipient, particularlywith allogeneic transplant and related Graft Versus Host Disease (GVHD)results. Therefore, the risk of GVHD restricts the use of bone marrowtransplantation to patients with otherwise fatal diseases. Further, asmost patients are elderly and only a few young MDS patients will have amatched donor, the use of bone marrow transplantation is limited. Thereremains a need for more effective methods for treating MDS and itsrelated disorders.

Mild MDS can grow more severe over time. It can also develop into afast-growing, severe leukemia, acute myelogenous leukemia (AML). AML isa subset of leukemia. It is the most common form of adult leukemia(blood cancer) with a <20% survival rate after 5 years and less than 5%if patients are age >65. Current treatments for AML are blunt and harshchemotherapies (i.e., cytarabine, anthracycliine, etc.) that are nottargeted and have limiting off-target toxicities. Thus, there is asevere unmet medical need for distinguishing patients who arepotentially sensitive to a certain treatment, and patients who are not.

The present invention meets this need and provides compositions andmethods for the effective treatment of cancers.

SUMMARY OF THE INVENTION

The present invention provides compositions and methods for treatingcancers. In some embodiments, the present invention is based, in part,on the discovery that the Caudal-related Homeobox proteinCDX2-Krüppel-like factor 4 (KLF4) signaling pathway (“the CDX-KLF4signaling pathway”) is important for the pathogenesis of a group ofcancers, and that active agents with an ability of modulating theCDX2-KLF4 signaling pathway can be used to treat such cancerseffectively.

In one aspect, the invention provides compositions for treating cancers.In some embodiments, the compositions comprise at least one anti-canceractive agent that can modulate the CDX2-KLF4 signaling pathway in ahuman subject. Without wishing to be bound by any particular theory, theanti-cancer agents of the present invention can act through one or moremechanisms. Such mechanisms include, but are not limited to: (1)inhibition of CDX2 activity; (2) induction of KLF4 activity; (3)induction of p21 CDK inhibitor; (4) induction of G1/S Cell cycle arrest;(5) induction of Caspase 3 enzyme; and (6) induction of apoptosis. Asused herein, the term “activity” of a component in the CDX2-KLF4signaling pathway can be a parameter at genomic DNA level,transcriptional level, post-transcriptional level, translational level,post-translational level, including, but not limited to gene activity,RNA activity, and protein activity. The gene activity can be gene copynumber, gene amplification number, or promoter activity, etc. RNAactivity can be mRNA abundance, synthesis rate, and/or stability, etc.Protein activity can be protein abundance, synthesis rate, stability,enzymatic activity, phosphorylation rate, modifications, bindingactivity, etc.

In some embodiments, the active agent is a small molecule pharmaceuticalcompound. In some embodiments, the pharmaceutical compound is a2,4,5-trisubstituted imidazole compound described in US 2007/0123553A1,or 2-indolyl imidazo[4,5-d]phenanthroline compounds described in U.S.Pat. No. 8,148,392, or functional derivatives thereof. In someembodiments, the pharmaceutical compound has the structure of formula I:

Wherein R1 is C1-C4 alkyl; and R2 is halogen.

In some embodiments, R¹ is methyl, isopropyl, or t-butyl.

In some embodiments, the pharmaceutical compound is selected from thegroup consisting of:

In some embodiments, the pharmaceutical compound has the structure offormula II:

In some embodiments, the cancer is associated with abnormal activity ofthe CDX2-KLF4 signaling pathway in the human subject. In someembodiments, one or more components in the CDX2-KLF4 signaling pathwayhave abnormal activity when compared to the activity of a control group.In some embodiments, the cancer is leukemia/lymphoma. In someembodiments, the cancer is myelodysplastic syndromes (MDS). In someembodiments, the MDS is high-risk MDS. In some embodiments, the canceris acute myelogenous leukemia (AML). In some embodiments, the AML isrefractory AML. In some embodiments, the AML is elderly AML. In someother embodiments, the cancer is acute lymphocytic leukemia (ALL). Insome embodiments, the ALL is a pediatric ALL. In some embodiments, thepediatric ALL is pediatric T-cell ALL or pediatric B-cell ALL. In someother embodiments, the cancer is chronic myelogenous leukemia (CML). Insome embodiments, the cancer is adult T-cell leukaemia (ATLL). In someembodiments, the cancer is caused by human T-lymphotrophic virus type 1.In some embodiments, the cancer is lymphoma, gastric cancer, or multiplemyeloma. In some embodiments, the lymphoma is Hodgkin lymphoma,non-Hodgkin's lymphoma (NHL), Burkitts lymphoma, or B-cell lymphoma. Insome embodiments, the cancer is a combination of any of the cancersdescribed herein.

In some embodiments, the human subject has one or more symptoms of MDS.In some embodiments, the human subject to be treated has an ineffectiveproduction of blood cells, such as myeloid blood cells. In someembodiments, the human subject has an anemia. In some embodiments, thehuman subject has low blood counts caused by bone marrow failure.

In some embodiments, the cancer is acute myelogenous leukemia (AML). Insome embodiments, the AML is refractory AML. In some embodiments, theAML is elderly AML. As used herein, elderly AML patients are thosepatients who have, or may have AML, with an age above 60. In someembodiments, elderly AML patients are in their first relapse. In someembodiments, the AML is not elderly AML. As used herein, non-elderly AMLpatients are those who have, or may have AML, with an age equal to orbelow 60. In some embodiments, the non-elderly AML patients are in theirsecond relapse.

In some embodiments, the cancer is acute lymphocytic leukemia (ALL). Insome embodiments, the ALL is a pediatric or child ALL. As used herein,pediatric or child ALL patients are those patients who have, or may haveALL, with an age under 21. In some embodiments, the ALL is not pediatricor child ALL. As used herein, non-pediatric ALL patients are those whohave, or may have ALL, with an age equal to or above 21.

In some embodiments, an anti-cancer compound of the present invention isadministered to a human subject in need as part of a combinationtherapy. In some embodiments, the combination therapy comprisesradiotherapy. In some embodiments, the combination therapy compriseschemotherapy.

In some embodiments, an effective amount of an anti-cancer agent or apharmacologically acceptable salt or solvate thereof of the presentinvention is administered to the human subject. In some embodiments, aneffective amount of the anti-cancer agent is effective to treat thecancer or symptoms of the cancer, effective to inhibit cancer cellproliferation, or effective to prevent or reduce severity of a futureoccurrence of cancer when administered to a subject who is susceptibleand/or who may develop a cancer or symptoms of the cancer. In someembodiments, the administration of the anti-cancer agent providesstatistically significant therapeutic effect or clinical efficacy fortreating the cancer. In some embodiments, an anti-cancer compound of thepresent invention is administered with a dosage of about 0.01 to about200 mg. In some embodiments, an anti-cancer compound of the presentinvention is administered with a dosage of about 0.05 to about 100 mg.In some embodiments, an anti-cancer compound of the present invention isadministered with a dosage of about 1.0 to about 50 mg. In someembodiments, the daily dosages of the compounds of the present inventiontypically fall within the range of about 0.01 to about 100 mg/kg of bodyweight, or within the range of about 20 mg/m2 to about 400 mg/m2 insingle or divided dose. In some embodiments, an anti-cancer compound ofthe present invention is administered once, twice, three times or moreper day.

In some embodiments, the treatment last 1, 2, 3, 4, 5, 6, 7, 8, 9, 10,or more cycles. In some embodiments, each cycle lasts at least 1, 2, 3,4, 5, 6, 7, 8, 9, 10 or more days. In some embodiments, the gap betweentwo cycles is at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more days.

In some embodiments, the anti-cancer agent is unit dosage form.

In some embodiments, the cancer to be treated is AML, the compound isLOR-253 (a.k.a. APTO-253), and the compound is administered with adosage of about 125 mg/m2. In certain embodiments, the compound isadministered two times per week for four weeks.

The invention provides methods for treating a condition associated withabnormal activity of the CDX2-KLF4 signaling pathway. In someembodiments, the condition is associated with abnormal activity of oneor more component of the CDX2-KLF4 signaling pathway, such as CDX2,KLF4, p21, and/or p53 in a human subject. In some embodiments, thecondition is associated with abnormal (e.g., lower than normal) level ofKLF4 activity. In some embodiments, the condition is associated withabnormal (e.g., higher than normal) level of CDX2 activity. In someembodiments, the abnormal level of CDX2 is due to a genetic alterationof CDX2.

In some embodiments, the methods of the present invention furthercomprise determining the activity of a component in the CDX2-KLF4signaling pathway, such as the KLF4 activity and/or CDX2 activity in thehuman subject before, during, and/or after the treatment. In someembodiments, the methods of the present invention further comprisedetermining the presence or absence of a genetic alteration of CDX2.

Additional aspects and embodiments of the invention will be apparentfrom the detailed description that follows.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a diagram of non-limiting mechanism of how an active agent ofthe present invention (e.g., formula I, a.k.a. LOR-253 or APTO-253)treats cancer. Without wishing to be bound by any particular theory, incertain cancer types, such as solid tumors, KLF4 is down regulated,which is essential for accelerated cell proliferation,epithelial-mesenchymal transition (EMT) and metastasis; in certainhematologic cancers: KLF4 is down regulated, which is essential forleukemogenesis. LOR-253 induces KLF4 expression which in turn inhibitscancer cell proliferation, EMT, and metastasis, and/or triggersapoptosis.

FIG. 2 is a diagram that shows non-limiting mechanism of how CDX2functions in certain cancers and how LOR-253 treats such cancers.Without wishing to be bound by any particular theory, in normal cells ofthe hematopoietic system, the CDX2 gene is turned off or expressed at arelatively low level. In certain cancers including AML, CDX2 is turnedon or increased, leading to aberrantly expressed CDX2 transcriptionfactor. CDX2 binds to the promoter region of the KLF4 gene and inhibitsKLF4 expression, which is an essential step to promote leukemogenesis.LOR-253 induces KLF4 expression which in turn triggers apoptosis of thecancerous leukocytes.

FIG. 3 is a graphical representation of the effect of LOR-253 onproliferation of a number of cancer cell lines in vitro. Cells ofvarious cancer cell lines were incubated with LOR-253 as described inthe Examples, and the cell concentrations of various cell lines for 50%of maximal inhibition of cell proliferation (GI₅₀) by LOR-253 weredetermined and shown in the figure.

FIG. 4 is a graphical representation of the effect of LOR-253 on theexpression levels of KLF4 in two cell lines, THP-1 and HL60.

FIG. 5 depicts BD FACSCalibur flow cytometer assay result for THP1 andHL-60 AML cell lines treated with DMSO or LOR-253 (left panel). FIG. 5also shows that treatment of LOR-253 results in G1/S cell cycle arrestin THP1 and HL-60 cell lines (right panel).

FIG. 6 depicts a BD FACSCanto flow cytometry acquisition plots for THP-1cells treated with DMSO, 0.5 or 1 uM of LOR-253. THP-1 cells treatedwith LOR-253 showed elevated Annexin V staining, indicating induction ofapoptosis (Q3: Annexin V+/PI−).

FIG. 7 depicts Caspase 3 expression level in THP1 and HL-60 AML celllines treated with DMSO or LOR-253.

FIG. 8 depicts fold change in expression of BAX and BCL2 in THP1 cellstreated with DMSO or 0.5 μM LOR-253

FIG. 9 depicts in vivo efficacy of LOR-253 HCL in H226 xenograft modelmice. Tumor sizes of H226 xenograft mice treated with LOR-253 HCL ornegative control measured on the indicated days are shown.

FIG. 10 depicts the pharmacokinetic (PK) in CD-1 nude mice treated byLOR-253 at a dosage of 1, 5, or 15 mg/kg. The serum level of LOR-253 hasa dose related increase.

FIG. 11 depicts the pharmacodynamic (PD) responses in mice treated for 5consecutive days with 1, 5, and 15 mg/kg LOR-253. The KLF4 protein levelwas measured 16 hours after the last dose.

FIG. 12 depicts tumor shrinkage in patient with NSCLC (poorlydifferentiated adenocarcinoma) before the treatment (see the upperpanel) and after the treatment (see the lower panel).

FIG. 13 is a diagram that shows non-limiting mechanism of how silencingKLF4 gene or gene product plays a central role in various hememalignancies. For example, epigenetic methylation of the KLF4 generelates to adult T-cell lymphoma patients, mutations in KLF4 genes orproteins relate to pediatric T-cell ALL patients, elevated microRNA-2909relates to pediatric B-cell ALL patients, aberrant expression of CDX2relates to AML, ALL, and MDS patients, which all lead to silencing ofKLF4 activity (including but not limited to expressional and functionalactivities). Silencing of KLF4 has also been observed in variouslymphoma. The lower part of the diagram illustrates that silenced KLF4causes increased cancer cell proliferation through various “cell fategenes”.

FIG. 14 is a diagram that shows non-limiting mechanism of how KLF4 geneactivity can be silenced by genetic mutations or epigenetic events andhow LOR-253/APTO-253 can induce KLF4 expression. Such epigenetic eventsinclude, but are not limited to, DNA hypomethylation or demethylation,aberrant/elevated expression of CDX2 which can lead to increasedpresence of a demethylase KDM5B in an upstream regulatory region of theklf4 gene, and elevated amount of miR-2909. LOR-253/APTO-253 can induceKLF4 expression by relieving gene silencing caused at least byCDX2/KDM5B and/or other mechanisms

FIG. 15 depicts in vivo efficacy of LOR-253 HCL in Kasumi-1 xenograftmodel mice. Tumor sizes of Kasumi-1 xenograft mice treated with LOR-253HCL or negative control measured on the indicated days are shown.

FIG. 16 depicts body weight measurements on the indicated days ofKasumi-1 tumor-bearing Mice treated with LOR-253 HCL or negativecontrol.

FIG. 17 depicts in vivo efficacy of LOR-253 HCL as a single agent or incombination with azacitidine in HL-60 xenograft model mice. Tumor sizesof HL-60 xenograft mice treated with the indicated conditions measuredon the indicated days are shown.

FIG. 18 and FIG. 19 depict the tumor sizes of individual animals at thebeginning (Day 1) and end (Day 19) of the study of FIG. 17,respectively.

FIG. 20 depicts in vivo efficacy of LOR-253 HCL in KG-1 xenograft modelmice. Tumor sizes of KG-1 xenograft mice treated with LOR-253 HCL ornegative control measured on the indicated days are shown.

FIG. 21 depicts in vivo efficacy of LOR-253 HCL as a single agent or incombination with azacitidine in THP-1 xenograft model mice. Tumor sizesof THP-1 xenograft mice treated with the indicated conditions measuredon the indicated days are shown.

DETAILED DESCRIPTION Definitions

The verb “comprise” as is used in this description and in the claims andits conjugations are used in its non-limiting sense to mean that itemsfollowing the word are included, but items not specifically mentionedare not excluded.

The term “a” or “an” refers to one or more of that entity; for example,“a gene” refers to one or more genes or at least one gene. As such, theterms “a” (or “an”), “one or more” and “at least one” are usedinterchangeably herein. In addition, reference to “an element” by theindefinite article “a” or “an” does not exclude the possibility thatmore than one of the elements are present, unless the context clearlyrequires that there is one and only one of the elements.

The invention provides isolated, chimeric, recombinant or syntheticpolynucleotide sequences. As used herein, the terms “polynucleotide”,“polynucleotide sequence”, “nucleic acid sequence”, “nucleic acidfragment”, and “isolated nucleic acid fragment” are used interchangeablyherein and encompass DNA, RNA, cDNA, whether single stranded or doublestranded, as well as chemical modifications thereof. These termsencompass nucleotide sequences and the like. A polynucleotide may be apolymer of RNA or DNA that is single- or double-stranded, thatoptionally contains synthetic, non-natural or altered nucleotide bases.A polynucleotide in the form of a polymer of DNA may be comprised of oneor more segments of cDNA, genomic DNA, synthetic DNA, or mixturesthereof. Nucleotides (usually found in their 5′-monophosphate form) arereferred to by a single letter designation as follows: “A” for adenylateor deoxyadenylate (for RNA or DNA, respectively), “C” for cytidylate ordeoxycytidylate, “G” for guanylate or deoxyguanylate, “U” for uridylate,“T” for deoxythymidylate, “R” for purines (A or G), “Y” for pyrimidines(C or T), “K” for G or T, “H” for A or C or T, “I” for inosine, and “N”for any nucleotide. In some embodiments, the isolated, chimeric,recombinant or synthetic polynucleotide sequences are derived from genemarkers of the present invention.

The invention also provides proteins or polypeptides. In someembodiments the proteins or polypeptides are isolated, purified,chimeric, recombinant or synthetic. As used herein, the term“polypeptide” or “protein” refers to amino acid polymers of any length.The polymer may be linear or branched, it may comprise modified aminoacids, and it may be interrupted by non-amino acids. The terms alsoencompass an amino acid polymer that has been modified naturally or byintervention; for example, disulfide bond formation, glycosylation,lipidation, acetylation, phosphorylation, or any other manipulation ormodification, such as conjugation with a labelling component. Alsoincluded are, for example, polypeptides containing one or more analogsof an amino acid (including, for example, unnatural amino acids, etc),as well as other modifications known in the art. Polypeptides can occuras single chains or associated chains. Polypeptides of the invention cantake various forms (e.g. native, fusions, glycosylated,non-glycosylated, lipidated, non-lipidated, phosphorylated,non-phosphorylated, myristoylated, non-myristoylated, monomeric,multimeric, particulate, denatured, etc). In some embodiments, thesequences of the proteins or polypeptides are derived from gene markersof the present invention.

Single letter amino acid abbreviations used herein have their standardmeaning in the art, and all peptide sequences described herein arewritten according to convention, with the N-terminal end to the left andthe C-terminal end to the right.

As used herein, the term “component in the CDX2-KLF4 signaling pathway”,refers to CDX2, KLF4, or other genes, gene products (including but notlimited to RNA and protein), or other biological molecules that canmodulate the activity of CDX2 and/or KLF4, directly or indirectly, orgenes, gene products, or other biological molecules that can bemodulated by CDX2 and/or KLF4, directly or indirectly. The modulationcan either increase or decrease the activity level of a given gene. Suchcomponents include, but are not limited to CDX2, KLF4, KDM5B, miR-2909,p53, p21, Caspase-3, Annexin V, BAX, BCL2, BCL3, BMP, Wnt, HNF4α, Fgf,Hox, SP1, MYC, CCND1, AATF, and those described in Scholl et al. (“Thehomeobox gene CDX2 is aberrantly expressed in most cases of acutemyeloid leukemia and promotes leukemogenesis”, J. Clin. Invest.117:1037-1048 (2007).), Yoon et al. (Krüppel-like Factor 4 Mediatesp53-dependent G1/S Cell Cycle Arrest in Response to DNA Damage, Vol.278, No. 4, Issue of January 24, pp. 2101-2105, 2003), Faber et al.(CDX2-driven leukemogenesis involves KLF4 repression and deregulatedPPARγ signaling, J Clin Invest. doi:10.1172/JCI64745.), Rouhi et al.(“Deregulation of the CDX2-KLF4 axis in acute myeloid leukemia and coloncancer”, Oncotarget. 2013 February; 4(2):174-175.), Lengerke et al.(“BMP and Wnt specify hematopoietic fate by activation of the Cdx-Hoxpathway”, Cell Stem Cell. 2008 Jan. 10; 2(1):72-82.), Saandi et al.(“Regulation of the tumor suppressor homeogene Cdx2 by HNF4α inintestinal cancer”, Oncogene. 2013 August 8; 32(32):3782-8.), Malik etal., (miR-2909-mediated regulation of KLF4: a novel molecular mechanismfor differentiating between B-cell and T-cell pediatric acutelymphoblastic leukemias. Mol Cancer. 13:175, 2014), and Rowland et al.(“KLF4, p21 and context-dependent opposing forces in cancer”, Nat RevCancer. 2006 January; 6(1):11-23), each of which is incorporated hereinby reference in its entirety for all purposes. KLF4 negatively regulates(or suppresses) the activity of SP1, MYC, BCL3, CCND1, and AATF,directly or indirectly, while positively regulates the activity of p21.In addition, KLF4 negatively regulates the activity of p53 in somecancer types (e.g. breast cancer, as described by Rowland et al., TheKLF4 tumour suppressor is a transcriptional repressor of p53 that actsas a context-dependent oncogene. Nat Cell Biol. 2005. 7:1074-82), butpositively regulates the activity of p53 in some other cancer types(e.g., colon cancer and multiple myeloma, as described by Ghaleb et al.(Krüppel-like factor 4 exhibits antiapoptotic activity followinggamma-radiation-induced DNA damage. Oncogene. 2007. 26:2365-73), andSchoenhals et al. (Krüppel-like factor 4 blocks tumor cell proliferationand promotes drug resistance in multiple myeloma. Haematologica. 2013.98:1442-9)). Many of the genes regulated by KLF4 are referred to as“cell fate genes”. An illustration of some possible effects of silencedKLF4 gene expression or activity on some of the genes modulated by KLF4can be found in FIG. 13. Some other components in the CDX2-KLF4signaling pathway can modulate the expression of KLF4. Examples of suchcomponents include, but are not limited to, CDX2, KDM5B (a demethylase),and a miRNA “miR-2909”. An illustration of some possible mechanisms ofhow these KLF4 modulators can affect KLF4 gene expression or activity invarious cancer types can be found in FIG. 13 and FIG. 14. A component inthe CDX2-KLF4 signaling pathway can be used as a biomarker according tothe methods described herein for treating cancers, especially fortreating cancers by an anti-cancer agent of the present invention, suchas LOR-253/APTO-253.

As used herein, the term “modulate the CDX2-KLF4 signaling pathway”refers to the process in which one or more component in the CDX2-KLF4signaling pathway is modulated by an agent or an event (including amutation). In some embodiments, such modulation leads to increased,decreased, normalized, and/or stabilized activity of one or morecomponents in the CDX2-KLF4 signaling pathway.

The term lower alkyl refers to (C₁-C₆)alkyl. A lower alkyl includesmethyl, ethyl, propyl, isopropyl, butyl, iso-butyl, sec-butyl, pentyl,3-pentyl, hexyl, (C₃-C₆)cycloalkyl (e.g., cyclopropyl, cyclobutyl,cyclopentyl, or cyclohexyl), (C₃-C₆)cycloalkyl(C₁-C₆)alkyl (e.g.,cyclopropylmethyl, cyclobutylmethyl, cyclopentylmethyl,cyclohexylmethyl, 2-cyclopropylethyl, 2-cyclobutylethyl,2-cyclopentylethyl, or 2-cyclohexylethyl), (C₁-C₆)alkoxy (e.g., methoxy,ethoxy, propoxy, isopropoxy, butoxy, iso-butoxy, sec-butoxy, pentoxy,3-pentoxy, or hexyloxy) (C₂-C₆)alkenyl (e.g., vinyl, allyl, 1-propenyl,2-propenyl, 1-butenyl, 2-butenyl, 3-butenyl, 1-pentenyl, 2-pentenyl,3-pentenyl, 4-pentenyl, 1-hexenyl, 2-hexenyl, 3-hexenyl, 4-hexenyl, or5-hexenyl), (C₂-C₆)alkynyl (e.g., ethynyl, 1-propynyl, 2-propynyl,1-butynyl, 2-butynyl, 3-butynyl, 1-pentynyl, 2-pentynyl, 3-pentynyl,4-pentynyl, 1-hexynyl, 2-hexynyl, 3-hexynyl, 4-hexynyl, or 5-hexynyl),(C₁-C₆)alkanoyl (e.g., acetyl, propanoyl or butanoyl), halo(C₁-C₆)alkyl(e.g., iodomethyl, bromomethyl, chloromethyl, fluoromethyl,trifluoromethyl, 2-chloroethyl, 2-fluoroethyl, 2,2,2-trifluoroethyl, orpentafluoroethyl), hydroxy(C₁-C₆)alkyl (e.g., hydroxymethyl,1-hydroxyethyl, 2-hydroxyethyl, 1-hydroxypropyl, 2-hydroxypropyl,3-hydroxypropyl, 1-hydroxy butyl, 4-hydroxybutyl, 1-hydroxypentyl,5-hydroxypentyl, 1-hydroxyhexyl, or 6-hydroxyhexyl),(C₁-C₆)alkoxycarbonyl (e.g., methoxycarbonyl, ethoxycarbonyl,propoxycarbonyl, isopropoxycarbonyl, butoxycarbonyl, pentoxycarbonyl, orhexyloxycarbonyl), (C₁-C₆)alkylthio (e.g., methylthio, ethylthio,propylthio, isopropylthio, butylthio, isobutylthio, pentylthio, orhexylthio), and/or (C₂-C₆)alkanoyloxy (e.g., acetoxy, propanoyloxy,butanoyloxy, isobutanoyloxy, pentanoyloxy, or hexanoyloxy).

A compound described herein or its function derivative can be usedaccording to the present invention. The term “derivative” as used hereinincludes derivatives, analogs, prodrugs, and unnatural precursors of agiven compound.

As used herein, the term “treatment efficacy” and variants thereof aregenerally indicated by alleviation of one or more signs or symptomsassociated with the disease and can be readily determined by one skilledin the art. “Treatment efficacy” may also refer to the prevention oramelioration of signs and symptoms of toxicities typically associatedwith standard or non-standard treatments of a disease. Determination oftreatment efficacy is usually indication and disease specific and caninclude any methods known or available in the art for determining that atreatment is providing a beneficial effect to a subject. For example,evidence of treatment efficacy can include but is not limited to generalimprovements in the overall health of the subject, such as but notlimited to enhancement of patient life quality, increase in predictedsubject survival rate, decrease in depression, decreasing the severityand/or frequency one or more symptoms resulting from the disease,diminishing the extent of the disease, stabilizing the disease (e.g.,preventing or delaying the worsening of the disease), delay or slowingthe progression of the disease, ameliorating the disease state, etc. Insome embodiments of the invention, the treatment efficacy is clinicalefficacy or statistically significant.

The terms “treating” and “treatment” as used herein refer to an approachfor obtaining beneficial or desired results including clinical results,and may include even minimal changes or improvements in one or moremeasurable markers of the disease or condition being treated. Atreatment is usually effective to reduce at least one symptom of acondition, disease, disorder, injury or damage. Exemplary markers ofclinical improvement will be apparent to persons skilled in the art.Examples include, but are not limited to, one or more of the following:decreasing the severity and/or frequency one or more symptoms resultingfrom the disease, diminishing the extent of the disease, stabilizing thedisease (e.g., preventing or delaying the worsening of the disease),delay or slowing the progression of the disease, ameliorating thedisease state, decreasing the dose of one or more other medicationsrequired to treat the disease, and/or increasing the quality of life,etc.

“Prophylaxis,” “prophylactic treatment,” or “preventive treatment”refers to preventing or reducing the occurrence or severity of one ormore symptoms and/or their underlying cause, for example, prevention ofa disease or condition in a subject susceptible to developing a diseaseor condition (e.g., at a higher risk, as a result of geneticpredisposition, environmental factors, predisposing diseases ordisorders, or the like).

The term “disorder” or “disease” used interchangeably herein, refers toany alteration in the state of the body or one of its organs and/ortissues, interrupting or disturbing the performance of organ functionand/or tissue function (e.g., causes organ dysfunction) and/or causing asymptom such as discomfort, dysfunction, distress, or even death to asubject afflicted with the disease.

By “pharmaceutically acceptable” is meant a material that is notbiologically or otherwise undesirable, i.e., the material may beincorporated into a pharmaceutical composition administered to a patientwithout causing any significant undesirable biological effects orinteracting in a deleterious manner with any of the other components ofthe composition in which it is contained. When the term“pharmaceutically acceptable” is used to refer to a pharmaceuticalcarrier or excipient, it is implied that the carrier or excipient hasmet the required standards of toxicological and manufacturing testing orthat it is included on the Inactive Ingredient Guide prepared by theU.S. Food and Drug administration.

The term “effective amount” refers to the amount of one or morecompounds that renders a desired treatment outcome. An effective amountmay be comprised within one or more doses, i.e., a single dose ormultiple doses may be required to achieve the desired treatmentendpoint.

The term “therapeutically effective amount” as used herein, refers tothe level or amount of one or more agents needed to treat a condition,or reduce or prevent injury or damage, optionally without causingsignificant negative or adverse side effects.

A “prophylactically effective amount” refers to an amount of an agentsufficient to prevent or reduce severity of a future disease orcondition when administered to a subject who is susceptible and/or whomay develop a disease or condition.

According to the methods of the present invention, the term “subject,”and variants thereof as used herein, includes any subject that has, issuspected of having, or is at risk for having a disease or condition.Suitable subjects (or patients) include mammals, such as laboratoryanimals (e.g., mouse, rat, rabbit, guinea pig), farm animals, anddomestic animals or pets (e.g., cat, dog). Non-human primates and,preferably, human patients, are included. A subject “at risk” may or maynot have detectable disease, and may or may not have displayeddetectable disease prior to the diagnostic or treatment methodsdescribed herein. “At risk” denotes that a subject has one or moreso-called risk factors, which are measurable parameters that correlatewith development of a condition described herein, which are describedherein. A subject having one or more of these risk factors has a higherprobability of developing a condition described herein than a subjectwithout these risk factor(s). One example of such a risk factor is anincrease or decrease in a biomarker of the present invention as comparedto a clinically normal sample.

In certain embodiments, when measuring the activity level of a componentin the CDX2-KLF4 signaling pathway of treatment, an “increased” or“decreased” amount or level may include a “statistically significant”amount. In some embodiments, the administration of an anti-cancer agentsuch as LOR-253 provides “statistically significant” therapeutic effector clinical efficacy for treating the cancer. In some embodiments, suchstatistically significant therapeutic effect or clinical efficacyincludes slower cancer cell proliferation or tumor growth caused by theanti-cancer agent as compared to a control vehicle. A result istypically referred to as statistically significant if it is unlikely tohave occurred by chance. The significance level of a test or resultrelates traditionally to the amount of evidence required to accept thatan event is unlikely to have arisen by chance. In certain cases,statistical significance may be defined as the probability of making adecision to reject the null hypothesis when the null hypothesis isactually true (a decision known as a Type I error, or “false positivedetermination”). This decision is often made using the p-value: if thep-value is less than the significance level, then the null hypothesis isrejected. The smaller the p-value, the more significant the result.Bayes factors may also be utilized to determine statistical significance(see, e.g., Goodman S., Ann Intern Med. 130:1005-13, 1999). In someembodiments, an “increased” or “decreased” amount or level is about1.1×, 1.2×, 1.3×, 1.4×, 1.5×, 2×, 2.5×, 3×, 3.5×, 4×, 4.5×, 5×, 6×, 7×,8×, 9×, 10×, 15×, 20×, 25×, 30×, 40×, or 50× more or less the amount ofa predetermined standard, or the amount of a determined time pointrelative to a previous or earlier timepoint.

According to some embodiments of the present invention, administering ofanti-cancer agents such as LOR-253 according to the methods of thepresent invention provides statistically significant therapeutic effect.In one embodiment, the statistically significant therapeutic effect isdetermined based on one or more standards or criteria provided by one ormore regulatory agencies in the United States, e.g., FDA or othercountries. In other embodiments, the statistically significanttherapeutic effect is determined based on results obtained fromregulatory agency approved clinical trial set up and/or procedure.

In some embodiments, the statistically significant therapeutic effect isdetermined based on a patient population of at least 300, 400, 500, 600,700, 800, 900, 1000 or 2000. In some embodiments, the statisticallysignificant therapeutic effect is determined based on data obtained fromrandomized and double blinded clinical trial set up. In someembodiments, the statistically significant therapeutic effect isdetermined based on data with a p value of less than or equal to about0.05, 0.04, 0.03, 0.02 or 0.01. In some embodiments, the statisticallysignificant therapeutic effect is determined based on data with aconfidence interval greater than or equal to 95%, 96%, 97%, 98% or 99%.In some embodiments, the statistically significant therapeutic effect isdetermined on approval of Phase III clinical trial of the methodsprovided by the present invention, e.g., by FDA in the US.

In some embodiments, the statistically significant therapeutic effect isdetermined by a randomized double blind clinical trial of a patientpopulation of at least 300 or 350, treated with anti-cancer agents suchas LOR-253 in combination with standard care. In some embodiment, thestatistically significant therapeutic effect is determined by arandomized clinical trial of a patient population of at least 300 or 350and using 28 day mortality rate, in-hospital mortality rate, ICUmortality rate, ICU duration, ICU free days, sequential organ failureassessment score (SOFA), relative risk of death, ICU frequency, durationof ventilation, frequency of ventilation, ventilation free days or anycombination thereof or any other commonly accepted criteria for sepsisassessment.

In general, statistical analysis can include any suitable methodpermitted by a regulatory agency, e.g., FDA in the US or China or anyother country. In some embodiments, statistical analysis includesnon-stratified analysis, log-rank analysis, e.g., from Kaplan-Meier,Jacobson-Truax, Gulliken-Lord-Novick, Edwards-Nunnally, Hageman-Arrindeland Hierarchical Linear Modeling (HLM) and Cox regression analysis.

As used herein, the phrase “per day” describes an amount administered onthose days when an agent is administered. The phrase “per day” does notindicate that an amount is administered every day.

The phrase “pharmaceutically acceptable salt(s)”, as used herein, unlessotherwise indicated, includes salts of acidic or basic groups which maybe present in a compound. Compounds that are basic in nature are capableof forming a wide variety of salts with various inorganic and organicacids. The acids that may be used to prepare pharmaceutically acceptableacid addition salts of such basic compounds are those that formnon-toxic acid addition salts, i.e., salts containing pharmacologicallyacceptable anions, such as the acetate, benzenesulfonate, benzoate,bicarbonate, bisulfate, bistosylate, bitartrate, borate, bromide,calcium edetate, camsylate, carbonate, chloride, clavulanate, citrate,dihydrochloride, edetate, edislyate, estolate, esylate, ethylsuccinate,fumarate, gluceptate, gluconate, glutamate, glycollylarsanilate,hexylresorcinate, hydrabamine, hydrobromide, hydrochloride, iodide,isothionate, lactate, lactobionate, laurate, malate, maleate, mandelate,mesylate, methylsulfate, mucate, napsylate, nitrate, oleate, oxalate,pamoate (embonate), palmitate, pantothenate, phosphate/diphosphate,polygalacturonate, salicylate, stearate, subacetate, succinate, tannate,tartrate, teoclate, tosylate, triethiodode, and valerate salts.

The term “prodrug”, as used herein, unless otherwise indicated, meanscompounds that are drug precursors, which following administration,release the drug in vivo via some chemical or physiological process(e.g., a prodrug on being brought to the physiological pH is convertedto the desired drug form).

“Continuous dosing schedule”, as used herein, unless otherwiseindicated, refers to a dosing schedule wherein compound of presentinvention, or a dosage form comprising said compound, is administeredduring a treatment period without a rest period. Throughout thetreatment period of a continuous dosing schedule, the compound, or adosage form comprising the compound, can be administered, for example,daily, every other day, every third day, every forth day, every fifthday, etc. On a day when compound, or a dosage form comprising thecompound is administered, it can be administered in a single dose, or inmultiple doses throughout the day.

“Intermittent dosing schedule”, as used herein, unless otherwiseindicated, refers to a dosing schedule that comprises a treatment periodand a rest period. Throughout the treatment period of an intermittentdosing schedule, compound of present invention, or a dosage formcomprising said compound, can be administered, for example, daily, orevery other day, every third day, every forth day, every fifth day, etc.On a day when compound or a dosage form comprising the compound isadministered, it can be administered in a single dose, or in multipledoses throughout the day. During the rest period, a compound or a dosageform comprising the compound is not administered. In some embodiments,the rest period lasts at least one day, at least two days, at leastthree days, at least four days, at least five days, at least six days,at least a week, at least 1.5 week, at least 2 weeks, at least 3 weeks,at least 4 weeks, at least one month, at least two months, at leastthree months, at least four months, at least five months, at least halfyear, at least one year, at least two years, or more. In someintermittent dosing regimens, the treatment period is typically fromabout 1 day to 30 days, such as about 10 days to 30 days, for example,about 2, 3 or 4 weeks, and the rest period is typically from 1 to 30days, such as 3 to 15 days, for example, 1 or 2 weeks. The combinationof any treatment period from 10 to 30 days with any rest period from 3to 15 days is contemplated. Intermittent dosing regimens can beexpressed as treatment period in weeks/rest period in weeks. Forexample, a 4/1 intermittent dosing schedule refers to an intermittentdosing schedule wherein the treatment period is four weeks and the restperiod is one week. A 4/2 intermittent dosing schedule refers to anintermittent dosing schedule wherein the treatment period is four weeksand the rest period is two weeks. Similarly, a 3/1 intermittent dosingschedule refers to an intermittent dosing schedule wherein the treatmentperiod is three weeks and the rest period is one week.

The human subject treated by the anti-cancer agent of the presentinvention may show complete response or partial response. CompleteResponse (CR), as used herein, unless otherwise indicated, refers todisappearance of all measurable and nonmeasurable symptoms and noappearance of new symptoms in a patient under the treatment. PartialResponse (PR), as used herein, unless other wise indicated, refers to atleast one measurable and nonmeasurable symptom is significantly reduced,or without appearance of new symptom in a patient under treatment.

It should be further appreciated that dosing regimens can be adjusted byone skilled in the art to more conveniently accommodate coordination ofthe dosing regimens, and additional therapeutic agents, if suchadjustments are therapeutically acceptable.

As used herein, “C_(max)” refers to the maximum plasma concentration;t_(max) refers to the time when the C_(max) occurs followingadministering the dosage; AUC refers to area under the plasmaconcentration-time curve from time zero to infinity; t_(1/2) refers toplasma elimination half-life; % CV refers to percent coefficient ofvariation; C_((trough 24 h)) refers to trough plasma concentration at 24hours after dosing; and QD indicates once daily.

Cancers

The cancers which can be treated in accordance with one embodiment ofthe present invention thus include, but are not limited to, leukaemias;adenocarcinomas and carcinomas, including squamous cell carcinomas.Carcinomas are also frequently referred to as “solid tumours,” asdescribed above, and examples of commonly occurring solid tumours thatcan be treated in accordance with the present invention include, but arenot limited to, anal cancer, bladder cancer, colon cancer, colorectalcancer, duodenal cancer, gastric (stomach) cancer, lung (non-small cell)cancer, oesophageal cancer, prostate cancer, rectal cancer and smallintestine cancer. Accordingly, one embodiment of the present inventionprovides for the use of a compound of Formula I in the treatment of acancer selected from the group of leukemia, bladder cancer, lung(non-small cell) cancer, prostate cancer and a cancer of the GI tract,wherein cancers of the GI tract include, but are not limited to, analcancer, colon cancer, colorectal cancer, duodenal cancer, gastric(stomach) cancer, oesophageal cancer, rectal cancer and small intestinecancer. As used herein, “C_(max)” refers to the maximum plasmaconcentration; t_(max) refers to the time when the C_(max) occursfollowing administering the dosage; AUC refers to area under the plasmaconcentration-time curve from time zero to infinity; t_(1/2) refers toplasma elimination half-life; % CV refers to percent coefficient ofvariation; C_((trough 24 h)) refers to trough plasma concentration at 24hours after dosing; and QD indicates once daily.

The term “leukaemia” or “leukemia” refers broadly to progressive,malignant diseases of the blood-forming organs. Leukaemia is typicallycharacterized by a distorted proliferation and development of leukocytesand their precursors in the blood and bone marrow but can also refer tomalignant diseases of other blood cells such as erythroleukaemia, whichaffects immature red blood cells. Leukaemia is generally clinicallyclassified on the basis of (1) the duration and character of thedisease—acute or chronic; (2) the type of cell involved—myeloid(myelogenous), lymphoid (lymphogenous) or monocytic, and (3) theincrease or non-increase in the number of abnormal cells in theblood—leukaemic or aleukaemic (subleukaemic). Leukaemia includes, forexample, acute leukaemia, chronic leukaemia, adult leukaemia,pediatric/child leukaemia, lymphocytic leukemia, myeloid leukemia, acutelymphocytic leukemia (ALL), acute nonlymphocytic leukaemia, chroniclymphocytic leukaemia, acute granulocytic leukaemia, chronicgranulocytic leukaemia, acute promyelocytic leukaemia, chronicmyelogenous leukemia (CML), T-cell leukaemia, B-cell leukaemia, adultT-cell leukaemia, pediatric T-cell ALL, pediatric B-cell ALL, aleukaemicleukaemia, aleukocythemic leukaemia, basophylic leukaemia, blast cellleukaemia, bovine leukaemia, chronic myelocytic leukaemia, leukaemiacutis, embryonal leukaemia, eosinophilic leukaemia, Gross' leukaemia,hairy-cell leukaemia, hemoblastic leukaemia, hemocytoblastic leukaemia,histiocytic leukaemia, stem cell leukaemia, acute monocytic leukaemia,leukopenic leukaemia, lymphatic leukaemia, lymphoblastic leukaemia,lymphocytic leukaemia, lymphogenous leukaemia, lymphoid leukaemia,lymphosarcoma cell leukaemia, mast cell leukaemia, megakaryocyticleukaemia, micromyeloblastic leukaemia, monocytic leukaemia,myeloblastic leukaemia, myelocytic leukaemia, myeloid granulocyticleukaemia, myelomonocytic leukaemia, Naegeli leukaemia, plasma cellleukaemia, plasmacytic leukaemia, promyelocytic leukaemia, Rieder cellleukaemia, Schilling's leukaemia, stem cell leukaemia, subleukaemicleukaemia, and undifferentiated cell leukaemia.

The term “carcinoma” refers to a malignant new growth made up ofepithelial cells tending to infiltrate the surrounding tissues and giverise to metastases. The term “carcinoma” also encompassesadenocarcinomas. Adenocarcinomas are carcinomas that originate in cellsthat make organs which have glandular (secretory) properties or thatoriginate in cells that line hollow viscera, such as thegastrointestinal tract or bronchial epithelia, and includeadenocarcinomas of the lung and prostate.

Methods of the present invention can be applied in the treatment ofearly stage cancers including early neoplasias that may be small, slowgrowing, localized and/or nonaggressive, for example, with the intent ofcuring the disease or causing regression of the cancer, as well as inthe treatment of intermediate stage and in the treatment of late stagecancers including advanced and/or metastatic and/or aggressiveneoplasias, for example, to slow the progression of the disease, toreduce metastasis or to increase the survival of the patient. Similarly,the combinations may be used in the treatment of low grade cancers,intermediate grade cancers and or high grade cancers.

Methods of the present invention can also be used in the treatment ofindolent cancers, recurrent cancers including locally recurrent,distantly recurrent and/or refractory cancers (i.e. cancers that havenot responded to treatment), metastatic cancers, locally advancedcancers and aggressive cancers. Thus, an “advanced” cancer includeslocally advanced cancer and metastatic cancer and refers to overtdisease in a patient, wherein such overt disease is not amenable to cureby local modalities of treatment, such as surgery or radiotherapy. Theterm “metastatic cancer” refers to cancer that has spread from one partof the body to another. Advanced cancers may also be unresectable, thatis, they have spread to surrounding tissue and cannot be surgicallyremoved.

Methods of the present invention can also be used in the treatment ofdrug resistant cancers, including multidrug resistant tumours. As isknown in the art, the resistance of cancer cells to chemotherapy is oneof the central problems in the management of cancer.

One skilled in the art will appreciate that many of these categories mayoverlap, for example, aggressive cancers are typically also metastatic.“Aggressive cancer,” as used herein, refers to a rapidly growing cancer.One skilled in the art will appreciate that for some cancers, such asbreast cancer or prostate cancer the term “aggressive cancer” will referto an advanced cancer that has relapsed within approximately the earliertwo-thirds of the spectrum of relapse times for a given cancer, whereasfor other types of cancer, nearly all cases present rapidly growingcancers which are considered to be aggressive. The term can thus cover asubsection of a certain cancer type or it may encompass all of othercancer types.

In some embodiments, the cancer is leukemia/lymphoma. In someembodiments, the cancer is acute myelogenous leukemia (AML). In someembodiments, the cancer is lymphoma, gastric cancer, multiple myeloma,myelodysplastic syndromes, or combinations thereof. In some otherembodiments, the cancer is T-cell leukemia, e.g., adult T-cell leukemiaassociated with epigenetic methylation of KLF4 gene. In some otherembodiments, the cancer is ALL, e.g., pediatric ALL. In some otherembodiments, the cancer is pediatric T-cell ALL associated with one ormore mutations in KLF4 gene or protein. In some other embodiments, thecancer is pediatric B-cell ALL associated with elevated miRNA-2902. Insome other embodiments, the cancer is AML, ALL or MDS, e.g., high riskMDS, all of which associated with higher than normal CDX2 activity. Insome other embodiments, the cancer is Hodgkins, Burkitts, or B-celllymphomas, all of which associated with methylation of KLF4 gene.

Acute myeloid leukemia (AML): also known as acute myelogenous leukemiaor acute nonlymphocytic leukemia (ANLL), is a cancer of the myeloid lineof blood cells, characterized by the rapid growth of abnormal whiteblood cells that accumulate in the bone marrow and interfere with theproduction of normal blood cells. The symptoms of AML are caused byreplacement of normal bone marrow with leukemic cells, which causes adrop in red blood cells, platelets, and normal white blood cells. Thesesymptoms include fatigue, shortness of breath, easy bruising andbleeding, and increased risk of infection. Several risk factors andchromosomal abnormalities have been identified, but the specific causeis not clear. As an acute leukemia, AML progresses rapidly and istypically fatal within weeks or months if left untreated. Several riskfactors for developing AML include, but are not limited to, preleukemicblood disorders, such as myelodysplastic syndrome or myeloproliferativedisease; exposure to anticancer chemotherapy; radiation, such as highamounts of ionizing radiation exposure; and genetic reasons, such asthose described in Taylor et al. (“The hereditary basis of humanleukemia”. In Henderson E S, Lister T A, Greaves M F. Leukemia (6thed.). Philadelphia: WB Saunders. p. 210. ISBN 0-7216-5381-2), Horwitz etal. (“Anticipation in familial leukemia”. Am. J. Hum. Genet. 59 (5):990-8. PMC 1914843. PMID 8900225), Crittenden (“An interpretation offamilial aggregation based on multiple genetic and environmentalfactors”. Ann. N. Y. Acad. Sci. 91 (3): 769-80), and Horwitz (“Thegenetics of familial leukemia”. Leukemia 11 (8): 1347-59). The WorldHealth Organization (WHO) classification of acute myeloid leukemiaattempts to be more clinically useful and to produce more meaningfulprognostic information than the FAB criteria. Each of the WHO categoriescontains numerous descriptive subcategories of interest to thehematopathologist and oncologist; however, most of the clinicallysignificant information in the WHO schema is communicated viacategorization into one of the subtypes listed below:

ICD-O (International Classification of Diseases Name Description forOncology) Acute myeloid Includes: Multiple leukemia with AML withtranslocations between recurrent chromosome 8 and 21 [t(8; 21)] genetic(ICD-O 9896/3); RUNX1/RUNX1T1 abnormalities AML with inversions inchromosome 16 [inv(16)] (ICD-O 9871/3); CBFB/MYH11 APL withtranslocations between chromosome 15 and 17 [t(15; 17)] (ICD-O 9866/3);RARA; PML AML with translocations in chromosomes 9 and 11 [t(9; 11)];MLLT3-MLL Patients with AML in this category generally have a high rateof remission and a better prognosis compared to other types of AML. AMLwith This category includes patients M9895/3 multilineage who have had aprior myelodys- dysplasia plastic syndrome (MDS) or myeloproliferativedisease (MPD) that transforms into AML. This category of AML occurs mostoften in elderly patients and often has a worse prognosis. AML and Thiscategory includes patients M9920/3 MDS, who have had prior chemotherapytherapy- and/or radiation and subsequently related develop AML or MDS.These leukemias may be characterized by specific chromosomalabnormalities, and often carry a worse prognosis. AML not Includessubtypes of AML that M9861/3 otherwise do not fall into the abovecategorized categories.

The French-American-British (FAB) classification system divides AML intoeight subtypes, M0 through to M7, based on the type of cell from whichthe leukemia developed and its degree of maturity. Although the WHOclassification (see above) may be more useful, the FAB system is stillwidely used.

Percentage of adult AML Type Name Cytogenetics patients M0 acutemyeloblastic leukemia,  5% minimally differentiated M1 acutemyeloblastic leukemia, 15% without maturation M2 acute myeloblasticleukemia, t(8; 21)(q22; q22), 25% with granulocytic maturation t(6; 9)M3 promyelocytic, or acute t(15; 17) 10% promyelocytic leukemia (APL) M4acute myelomonocytic leukemia inv(16)(p13q22), 20% del(16q) M4eomyelomonocytic together with inv(16), t(16; 16)  5% bone marroweosinophilia M5 acute monoblastic leukemia del (11q), t(9; 11), 10%(M5a) or acute monocytic t(11; 19) leukemia (M5b) M6 acute erythroidleukemias,  5% including erythroleukemia (M6a) and very rare pureerythroid leukemia (M6b) M7 acute megakaryoblastic t(1; 22)  5% leukemia

Previous methods for treating AML are described in Bishop J (“Thetreatment of adult acute myeloid leukemia”. Semin Oncol 24 (1): 57-69.1997), Weick et al. (“A randomized investigation of high-dose versusstandard-dose cytosine arabinoside with daunorubicin in patients withpreviously untreated acute myeloid leukemia: a Southwest Oncology Groupstudy” (PDF). Blood 88 (8): 2841-51, 1996), Bishop et al. (“A randomizedstudy of high-dose cytarabine in induction in acute myeloid leukemia”Blood 87 (5): 1710-7, 1996), Huang et al. (“Use of all-trans retinoicacid in the treatment of acute promyelocytic leukemia”. Blood 72 (2):567-72, 1988), Tallman et al. (“All-trans-retinoic acid in acutepromyelocytic leukemia”. N. Engl. J. Med. 337 (15): 1021-8, 1997),Fenaux et al. (“A randomized comparison of all transretinoic acid (ATRA)followed by chemotherapy and ATRA plus chemotherapy and the role ofmaintenance therapy in newly diagnosed acute promyelocytic leukemia. TheEuropean APL Group”. Blood 94 (4): 1192-200, 1999), Estey E (“Treatmentof acute myelogenous leukemia”. Oncology (Williston Park) 16 (3):343-52, 355-6; discussion 357, 362, 365-6, 2002), and Cassileth et al.(“Maintenance chemotherapy prolongs remission duration in adult acutenonlymphocytic leukemia”. J Clin Oncol 6 (4): 583-7, 1988).

Acute lymphocytic leukemia (ALL): also known as acute lymphoblasticleukemia, is an acute leukemia that is characterized by dysregulatedproliferation and accumulation of leukemic lymphocytes/lymphoblasts(immature white blood cells such as early B- and T-lymphocyteprogenitors) in the bone marrow and various extramedullary sites. ALL isthe most common type of cancer in children, and it is a relativelyuncommon cancer in adults. Risk factors for developing ALL include, butare not limited to, genetic disorders/mutations and various epigeneticmodifications such as those described by Iacobucci et al. (“Cytogeneticand molecular predictors of outcome in acute lymphocytic leukemia:recent developments”, Curr Hematol Malig Rep. 2012 June; 7(2):133-43.)and Florean et al. (“Epigenomics of leukemia: from mechanisms totherapeutic applications”. Epigenomics. 2011 October; 3(5):581-609).

Chronic myelogenous leukemia (CML): also known as chronic myeloidleukemia, is a chronic leukemia that is characterized bydysregulated/increased proliferation of various blood cells,predominantly myeloid cells in peripheral blood, and their precursors inthe bone marrow, resulting in their accumulation in the blood. It isless frequent than chronic lymphocytic leukemia (CLL) in adults in theWestern world, and the median age of CML onset is 50-60 years. Riskfactors for developing CML also include, but are not limited to, geneticdisorders/mutations and various epigenetic modifications such as thosedescribed by Florean et al. The disease course is triphasic, startingwith an early phase, also known as chronic phase (CP) disease. Then,leukemia stem cells can acquire additional genetic defects.

Adult T-cell leukaemia (ATLL): also known as adult T-cell lymphoma, isan uncommon lymphoproliferative disorder of mature CD4+ T cells that iscaused by the retrovirus human T-lymphotrophic virus type 1 (HTLV-1) asreviewed by Qayyum et al. (“Adult T-cell leukemia/lymphoma”. Arch PatholLab Med. 2014 February; 138(2):282-6). Presently, about 20 millionpeople worldwide are HTLV-1 carriers, with most infected individualsresiding in endemic areas such as southern Japan, Africa, the Caribbeanbasin, and Latin America. The lifelong viral carrier state and longlatency (20-40 years) are common after HTLV-1 infection, therefore thistype of leukemia/lymphoma is almost exclusively found in adults and isextremely rare in children. The lifetime risk of progression to ATLL inan HTLV-1-positive patient is 2.1% for women and 6.6% for men. The meanage of onset is 60 years old (range, 20-80 years old). The overwhelmingmajority of ATLL cases occur in patients infected during the early yearsof life, presumably because of a less efficient immune response in thisage group. In addition, the prolonged infection may increase chances ofaccruing subsequent mutations, and ultimately malignant transformation.Major paths of viral transmission are breast feeding, blood exposure,and unprotected sex. The World Health Organization Classification ofTumors of Hematopoietic and Lymphoid Tissues in 2008 subclassified ATLLinto 4 distinct variants according to the Shimoyama classification:acute (60%), lymphomatous (20%), chronic (15%), and smoldering (5%).There are no absolutely necessary features for each variant, and overlapis seen. The acute variant manifests as marked leukocytosis withatypical lymphocytes and eosinophilia. Symptoms include hypercalcemiawith or without osteolytic lesions, renal dysfunction andneuropsychiatric disturbances, elevated lactate dehydrogenase level,central nervous ring-enhancing lesions, and secondary respiratorycomplications. The lymphomatous variant is an aggressive advanceddisease resembling acute-onset subtype, and marked lymphadenopathywithout leukemia is a prominent feature of this variant. The chronicvariant typically presents with skin rash, leukocytosis with absolutelymphocytosis, mild lymphadenopathy, and hypercalcemia. The smolderingvariant is asymptomatic and is characterized by normal white blood cellcount with less than 5% circulating atypical lymphoid cells and withoutassociated hypercalcemia or organomegaly, although skin and pulmonaryinvolvement often occur. Progression from the smoldering variant to theacute variant can occur.

Lymphoma: Lymphoma is a type of blood cancer that occurs when B or Tlymphocytes, the white blood cells that form a part of the immune systemand help protect the body from infection and disease, divide faster thannormal cells or live longer than they are supposed to. Typically,lymphoma presents as a solid tumor of lymphoid cells. The current WHOclassification, published in 2001 and updated in 2008, is the latestclassification of lymphoma and is based upon the foundations laid withinthe “Revised European-American Lymphoma classification” (REAL):

A. Mature B-cell neoplasms:

-   -   Chronic lymphocytic leukemia/Small lymphocytic lymphoma    -   B-cell prolymphocytic leukemia    -   Lymphoplasmacytic lymphoma (such as Waldenström        macroglobulinemia)    -   Splenic marginal zone lymphoma    -   Plasma cell neoplasms:        -   Plasma cell myeloma        -   Plasmacytoma        -   Monoclonal immunoglobulin deposition diseases        -   Heavy chain diseases    -   Extranodal marginal zone B cell lymphoma, also called MALT        lymphoma    -   Nodal marginal zone B cell lymphoma (NMZL)    -   Follicular lymphoma    -   Mantle cell lymphoma    -   Diffuse large B cell lymphoma    -   Mediastinal (thymic) large B cell lymphoma    -   Intravascular large B cell lymphoma    -   Primary effusion lymphoma    -   Burkitt lymphoma/leukemia

B. Mature T cell and natural killer (NK) cell neoplasms

-   -   T cell prolymphocytic leukemia    -   T cell large granular lymphocytic leukemia    -   Aggressive NK cell leukemia    -   Adult T cell leukemia/lymphoma    -   Extranodal NK/T cell lymphoma, nasal type    -   Enteropathy-type T cell lymphoma    -   Hepatosplenic T cell lymphoma    -   Blastic NK cell lymphoma    -   Mycosis fungoides/Sezary syndrome    -   Primary cutaneous CD30-positive T cell lymphoproliferative        disorders        -   Primary cutaneous anaplastic large cell lymphoma        -   Lymphomatoid papulosis    -   Angioimmunoblastic T cell lymphoma    -   Peripheral T cell lymphoma, unspecified    -   Anaplastic large cell lymphoma

C. Hodgkin Lymphoma

-   -   Classical Hodgkin lymphomas:        -   Nodular sclerosis        -   Mixed cellularity        -   Lymphocyte-rich        -   Lymphocyte depleted or not depleted    -   Nodular lymphocyte-predominant Hodgkin lymphoma

D. Immunodeficiency-associated lymphoproliferative disorders

-   -   Associated with a primary immune disorder    -   Associated with the Human Immunodeficiency Virus (HIV)    -   Post-transplant    -   Associated with methotrexate therapy    -   Primary central nervous system lymphoma occurs most often in        immuno-compromised patients, in particular those with AIDS, but        it can occur in the immunocompetent as well. It has a poor        prognosis, particularly in those with AIDS. Treatment can        consist of corticosteroids, radiotherapy, and chemotherapy,        often with methotrexate.        Subtypes of lymphoma with relative incidence, histopathology,        immunophenotype, overall t-year survival are show below (Robbins        basic pathology (8th ed.). Philadelphia:        Saunders/Elsevier. 2007. pp. Table 12-8.):

Overall 5-year Lymphoma type Relative incidence ^([13]) Histopathology^([13]) Immunophenotype survival Other comments Precursor T-cell 40% ofLymphoblasts TdT, CD2, CD7 It often presents as a leukemia/lymphomalymphomas with irregular mediastinal mass in nuclear contours, becauseof childhood. condensed involvement of the chromatin, small thymus. Itis highly nucleoli and scant associated with cytoplasm NOTCH 1mutations. without granules. Most common in adolescent males. Follicular40% of Small “cleaved” CD10, surface Ig 72-77% Occurs in older lymphomalymphomas cells (centrocytes) adults. Usually in adults mixed with largeinvolves lymph activated cells nodes, bone marrow (centroblasts). andspleen. Usually nodular Associated with (“follicular”) t(14; 18)translocation growth pattern overexpressing Bcl-2. Indolent Diffuselarge B cell 40 to 50% Variable. Most Variable expression 60% Occurs inall ages, but lymphoma of resemble B cells of CD10 and most commonly inlymphomas of large germinal surface Ig older adults. Often in adultscenters. Diffuse occurs outside lymph growth pattern. nodes. Aggressive.Mantle cell 3 to 4% of Lymphocytes of CD5 50% to Occurs mainly inlymphoma lymphomas small to 70% adult males. Usually in adultsintermediate size involves lymph growing in nodes, bone marrow, diffusepattern spleen and GI tract. Associated with t(11; 14) translocationoverexpressing cyclin D1. Moderately aggressive. B-cell chronic 3 to 4%of Small resting CD5, surface 50% Occurs in older lymphocytic lymphomaslymphocytes immunoglobulin adults. Usually leukemia/lymphoma in adultsmixed with involves lymph variable number nodes, bone marrow of largeactivated and spleen. Most cells. Lymph patients have nodes areperipheral blood diffusely effaced involvement. Indolent. MALT lymphoma~5% of Variable cell size CD5, CD10, Frequently occurs lymphomas andsurface Ig outside lymph nodes. in adults differentiation. Veryindolent. May 40% show plasma be cured by local cell excision.differentiation. Homing of B cells to epithelium createslymphoepithelial lesions. Burkitt's lymphoma <1% of Round lymphoid CD10,surface Ig 50% Endemic in Africa, lymphomas cells of sporadic elsewhere.in the intermediate size More common in United with severalimmunocompromised States nucleoli. Starry- and in children. Often skyappearance visceral involvement. by diffuse spread Highly aggressive.with interspersed apoptosis. Mycosis fungoides Most Usually small CD475% Localized or more common lymphoid cells generalized skin cutaneouswith convoluted symptoms. Generally lymphoid nuclei that often indolent.In a more malignancy infiltrate the aggressive variant, epidermis,Sézary's disease, there creating Pautrier is skin erythema andmicroabscesseses. peripheral blood involvement. Peripheral T-cell MostVariable. Usually CD3 Probably consists of lymphoma-Not- common T a mixsmall to several rare tumor Otherwise-Specified cell large lymphoidtypes. It is often lymphoma cells with disseminated and irregularnuclear generally aggressive. contours. Nodular sclerosis MostReed-Sternberg CD15, CD30 Most common in form of Hodgkin common cellvariants and young adults. It often lymphoma type of inflammation.arises in the Hodgkin's usually broad mediastinum or lymphoma scleroticbands cervical lymph nodes. that consists of collagen. Mixed-cellularitySecond Many classic CD15, CD30 Most common in subtype of Hodgkin mostReed-Sternberg men. More likely to lymphoma common cells and bediagnosed at form of inflammation advanced stages than Hodgkin's thenodular sclerosis lymphoma form. Epstein-Barr virus involved in 70% ofcases.

Gastric cancer: a.k.a. stomach cancer, which refers to cancer arisingfrom any part of the stomach. Stomach cancer is often eitherasymptomatic (producing no noticeable symptoms) or it may cause onlynonspecific symptoms (symptoms which are not specific to just stomachcancer, but also to other related or unrelated disorders) in its earlystages. It can be diagnosed by gastroscopic exam, upper GI series, orcomputed tomography or CT scanning. It is previously treated by surgery,chemotherapy, and radiation.

Colorectal cancer: a.k.a. colon cancer, rectal cancer, bowel cancer orcolorectal adenocarcinoma, is a cancer from uncontrolled cell growth inthe colon or rectum (parts of the large intestine), or in the appendix.Greater than 75-95% of colon cancer occurs in people with little or nogenetic risk. Other risk factors include older age, male gender, highintake of fat, alcohol or red meat, obesity, smoking and a lack ofphysical exercise. Approximately 10% of cases are linked to insufficientactivity. The risk for alcohol appears to increase at greater than onedrink per day. Colorectal cancer is a disease originating from theepithelial cells lining the colon or rectum of the gastrointestinaltract, most frequently as a result of mutations in the Wnt signalingpathway that artificially increase signaling activity. The mutations canbe inherited or are acquired, and most probably occur in the intestinalcrypt stem cell. Genes in the Wnt signaling pathway that are related tocolorectal cancer include, but are not limited to, APC, β-catenin,AXIN1, AXIN2, TCF7L2, or NKD1. Beyond the defects in theWnt-APC-beta-catenin signaling pathway, other mutations must occur forthe cell to become cancerous. The p53 protein, produced by the TP53gene, normally monitors cell division and kills cells if they have Wntpathway defects. Eventually, a cell line acquires a mutation in the TP53gene and transforms the tissue from an adenoma into an invasivecarcinoma. Other apoptotic proteins commonly deactivated in colorectalcancers are TGF-β and DCC. Other oncogenes overexpressed in colorectalcancer include, genes encoding the proteins KRAS, RAF, and PI3K, whichnormally stimulate the cell to divide in response to growth factors, canacquire mutations that result in over-activation of cell proliferation.In addition to the oncogenic and inactivating mutations described forthe genes above, non-hypermutated samples also contain mutated CTNNB1,FAM123B, SOX9, ATM, and ARID1A. Progressing through a distinct set ofgenetic events, hypermutated tumors display mutated forms of ACVR2A,TGFBR2, MSH3, MSH6, SLC9A9, TCF7L2, and BRAF. The common theme amongthese genes, across both tumor types, is their involvement in WNT andTGF-β signaling pathways, which in turn results in increased activity ofMYC, a central player in colorectal cancer.

Multiple myeloma: a.k.a. plasma cell myeloma or Kahler's disease is acancer of plasma cells, a type of white blood cell normally responsiblefor producing antibodies. It can be symptomatic myeloma, asymptomaticmyeloma and MGUS (monoclonal gammopathy of undetermined significance).Myeloma is diagnosed with blood tests (serum protein electrophoresis,serum free kappa/lambda light chain assay), bone marrow examination,urine protein electrophoresis, and X-rays of commonly involved bones. Itis previously treated by steroids, chemotherapy, proteasome inhibitors,immunomodulatory drugs (IMiDs) such as thalidomide or lenalidomide, andstem cell transplants.

Myelodysplastic syndromes (MDS): Myelodysplastic syndrome (“MDS”) refersto a diverse group of hematopoietic stem cell disorders, which arehematological (blood-related) medical conditions with ineffectiveproduction (or dysplasia) of the myeloid class of blood cells. MDS ischaracterized by a cellular marrow with impaired morphology andmaturation (dysmyelopoiesis), peripheral blood cytopenias, and avariable risk of progression to acute leukemia, resulting fromineffective blood cell production. The Merck Manual 953 (17^(th) ed.1999) and List et al., 1990, J. Clin. Oncol. 8:1424. Some types of MDS,referred to as “low-risk MDS”, progress slowly and may cause mild tomoderate anemia, or decrements to other types of cells. Some other typesof MDS are called “high-risk MDS” and may cause severe problems. Inpatients with high-risk MDS, immature cells called blast cells make upmore than five percent of the cells in the marrow and do not developinto normal red cells, white cells and platelets, often causing moresevere deficiency in those cells/platelets. When MDS patients developmore than 20 percent blast cells, they are reclassified as having AMLwith trilineage dysplasia (AML-TLD).

The initial hematopoietic stem cell injury can be from causes such as,but not limited to, cytotoxic chemotherapy, radiation, virus, chemicalexposure, and genetic predisposition. A clonal mutation predominatesover bone marrow, suppressing healthy stem cells. In the early stages ofMDS, the main cause of cytopenias is increased programmed cell death(apoptosis). As the disease progresses and converts into leukemia, genemutation rarely occurs and a proliferation of leukemic cells overwhelmsthe healthy marrow. The disease course differs, with some cases behavingas an indolent disease and others behaving aggressively with a veryshort clinical course that converts into an acute form of leukemia.Patients with MDS can develop severe anemia and require bloodtransfusions. In some cases, the disease worsens and the patientdevelops cytopenias (low blood counts) caused by progressive bone marrowfailure.

According to French-American-British classification published in 1976,which was revised in 1982, cases were classified into five categories:

ICD-O Name Description M9980/3 Refractory anemia characterized by lessthan 5% (RA) primitive blood cells (myeloblasts) in the bone marrow andpathological abnormalities primarily seen in red cell precursors M9982/3Refractory anemia also characterized by less than 5% with ringmyeloblasts in the bone marrow, but sideroblasts distinguished by thepresence of 15% (RARS) or greater red cell precursors in the marrowbeing abnormal iron-stuffed cells called “ringed sideroblasts” M9983/3Refractory anemia characterized by 5-20% myeloblasts with excess blastsin the marrow (RAEB) M9984/3 Refractory anemia characterized by 21-30%myeloblasts with excess blasts in the marrow (>30% blasts is intransformation defined as acute myeloid leukemia) (RAEB-T) M9945/3Chronic myelomono- characterized by less than 20% cytic leukemiamyeloblasts in the bone marrow and (CMML), not to be greater than1*109/L monocytes (a confused with type of white blood cell) circulatingchronic myelogenous in the peripheral blood. leukemia or CML

The World Health Organization (WHO) modified this classification,introducing several new disease categories and eliminating others. Mostrecently the WHO has evolved a new classification scheme (2008) which isbased more on genetic findings:

Old system New system Refractory anemia (RA) Refractory cytopenia withunilineage dysplasia (Refractory anemia, Refractory neutropenia, andRefractory thrombocytopenia) Refractory anemia with Refractory anemiawith ring sideroblasts (RARS) ringed sideroblasts Refractory anemia withring sideroblasts - thrombocytosis (RARS-t) (provisional entity) (RARS)which is in essence a myelodysplastic/myeloproliferative disorder andusually has a JAK2 mutation (janus kinase) - New WHO classification 2008Refractory cytopenia with multilineage dysplasia (RCMD) includes thesubset Refractory cytopenia with multilineage dysplasia and ringsideroblasts (RCMD-RS). RCMD includes patients with pathological changesnot restricted to red cells (i.e., prominent white cell precursor andplatelet precursor (megakaryocyte) dysplasia. Refractory anemia withRefractory anemia with excess blasts I and II. RAEB was divided into*RAEB-I (5-9% excess blasts (RAEB) blasts) and RAEB-II (10-19%) blasts,which has a poorer prognosis than RAEB-I. Auer rods may be seen inRAEB-II which may be difficult to distinguish from acute myeloidleukemia. Refractory anemia with The category of RAEB-T was eliminated;such patients are now considered to have excess blasts in acuteleukemia. 5q- syndrome, typically seen in older women with normal orhigh transformation (RAEB-T) platelet counts and isolated deletions ofthe long arm of chromosome 5 in bone marrow cells, was added to theclassification. Chronic myelomonocytic CMML was removed from themyelodysplastic syndromes and put in a new category of leukemia (CMML)myelodysplastic-myeloproliferative overlap syndromes. 5q- syndromeMyelodysplasia unclassifiable (seen in those cases of megakaryocytedysplasia with fibrosis and others) Refractory cytopenia of childhood(dysplasia in childhood) - New WHO classification 2008

Signs and symptoms of MDS include, but are not limited to, anemia (lowred blood cell count or reduced hemoglobin), with chronic tiredness,shortness of breath, chilled sensation, sometimes chest pain; weaknessor feeling tired, paler skin, easy bruising or bleeding, petechiae,fever, neutropenia (low neutrophil count), with increased susceptibilityto infection; thrombocytopenia (low platelet count), with increasedsusceptibility to bleeding and ecchymosis (bruising), as well assubcutaneous hemorrhaging resulting in purpura or petechia; splenomegalyor rarely hepatomegaly; abnormal granules in cells, abnormal nuclearshape and size; and/or chromosomal abnormalities, including chromosomaltranslocations and abnormal chromosome number.

Many factors can increase risk of MDS, which include, but are notlimited to, being male or white, being older than 60 years, pasttreatment with chemotherapy or radiation therapy, being exposed tocertain chemicals, including tobacco smoke, pesticides, and solventssuch as benzene, and being exposed to heavy metals, such as mercury orlead.

Previous treatment methods for MDS include bone marrow transplantation,use of hematopoietic growth factors or cytokines to stimulate blood celldevelopment in a recipient, such as erythropoietin (EPO), granulocytemacrophage colony stimulating factor (GM-CSF), and granulocyte colonystimulating factor (G-CSF) (Metcalf, 1985, Science 229:16; Dexter, 1987,J. Cell Sci. 88:1; Golde and Gasson, 1988, Scientific American, July:62; Tabbara and Robinson, 1991, Anti-Cancer Res. 11:81; Ogawa, 1989,Environ. Health Presp. 80:199; and Dexter, 1989, Br. Med. Bull.45:337.). Unfortunately, bone transplantation is painful for donor andrecipient, and hematopoietic growth factors have not proven effective inmany clinical settings. Other methods include 5-azacytidine, decitabine,lenalidomide, immunosuppression, leukemia therapy, and investigationalapproaches.

In some embodiments, the histology of the cancer is determined before,during or after the treatment. Any suitable test can be used todetermine the histology of the cancer. Such test and examinationinclude, but are not limited to, common signs and symptoms of esophagealcancer, including but not limited to, backwards movement of food throughthe esophagus and possibly mouth (regurgitation), chest pain not relatedto eating, difficulty swallowing solids or liquids, heartburn, vomitingblood, hoarseness, chronic cough, hiccups, pneumonia, bone pain,bleeding into the esophagus, and weight loss, medical history andphysical exam, imaging tests, chest X-ray, computed tomography (CT)scan, magnetic resonance imaging (MRI) scan, positron emissiontomography (PET) scan, bone scan, sputum cytology, needle biopsy,bronchoscopy, endobronchial ultrasound, endoscopic esophagealultrasound, mediastinoscopy and mediastinotomy, thoracentesis,thoracoscopy, immunohistochemistry, molecular tests, blood tests, bariumswallow, endoscopic ultrasound, esophaogastroduodenoscopy (EGD) andbiopsy, or any suitable methods derived from thereof.

CDX2-KLF4 Signaling Pathway

The term “CDX2-KLF4 signaling pathway” as used herein refers to a groupof biological molecules that work together to control one or morecellular functions through CDX2 and/or KLF4, or by affecting theexpression or activity of CDX2 or KLF4 as described herein, directly orindirectly. Sometimes the expression level and/or activity of CDX2and/or KLF4 is also referred to as “CDX2-KLF4 axis”.

CDX2, a.k.a. Caudal Type Homeobox 2, CDX3, Caudal Type HomeoBoxTranscription Factor 2, Caudal-Type Homeobox Protein 2, or HomeoboxProtein CDX-2, is a member of the caudal-related homeobox transcriptionfactor gene family. The encoded protein is a major regulator ofintestine-specific genes involved in cell growth and differentiation.This protein also plays a role in early embryonic development of theintestinal tract. Aberrant expression of this gene is associated withintestinal inflammation and tumorigenesis. Diseases associated with CDX2include atrophic gastritis, and signet ring cell adenocarcinoma, andamong its related super-pathways are Transcription Role of VDR inregulation of genes involved in osteoporosis and Cytoskeleton remodelingRegulation of actin cytoskeleton by Rho GTPases. GO annotations relatedto this gene include transcription regulatory region sequence-specificDNA binding and sequence-specific DNA binding transcription factoractivity. An important paralog of this gene is CDX1. It is involved inthe transcriptional regulation of multiple genes expressed in theintestinal epithelium, and important in broad range of functions fromearly differentiation to maintenance of the intestinal epithelial liningof both the small and large intestine. DNA and protein sequences ofhuman CDX2 were previously reported, see GenBank Nos. NC_000013.10,NC_018924.2, NT_024524.14, NP_001256.3, ENSP00000370408, and Uniprot No.Q99626, each of which is incorporated herein by reference in itsentirety for all purposes. Such sequences can be utilized to designprocedures for detection and analysis of the level of CDX2 activity byways known to one skilled in the art. CDX2 is aberrantly expressed inmost cases of acute myeloid leukemia and promotes leukemogenesis (Schollet al., The Journal of Clinical Investigation, 17(4): 1037-1048), withan mRNA copy numbers varied between about 30 copies to about 89,000copies. As used herein, the phrase “CDX2 gene is turned on” or “CDX2activity is on” refers to that the mRNA copies of CDX2 in a humansubject is at least about 30 copies. Otherwise, the CDX2 gene isconsidered as being turned off.

Krüppel-Like Factor 4 (KLF4), a.k.a. Gut, EZF, GKLF, Epithelial ZincFinger Protein EZF, Gut-Enriched Krueppel-Like Factor, EndothelialKrüppel-Like Zinc Finger Protein, or Krueppel-Like Factor 4, isassociated with diseases including, but are not limited to, leukemia,skin squamous cell carcinoma, and familial adenomatous polyposis. Animportant paralog of this gene is KLF1. KLF4 can act both as activatorand as repressor. It binds the 5′-CACCC-3′ core sequence, such as thepromoter region of its own gene. It regulates the expression of keytranscription factors during embryonic development, and plays animportant role in maintaining embryonic stem cells, and in preventingtheir differentiation. It is required for establishing the barrierfunction of the skin and for postnatal maturation and maintenance of theocular surface. It is also involved in the differentiation of epithelialcells and may also function in skeletal and kidney development. Itfurther contributes to the down-regulation of p53/TP53 transcription,and induction of p21. DNA and protein sequences of human KLF4 werepreviously reported, see GenBank Nos. NC_000009.11, NT_008470.19,NC_018920.2, and Uniprot No. 043474, each of which is incorporatedherein by reference in its entirety for all purposes. Such sequences canbe utilized to design procedures for detection and analysis of the levelof KLF4 activity by ways known to one skilled in the art.

p21, a.k.a. Cyclin-Dependent Kinase Inhibitor 1A, Cip1, CDJN1, CIP1,WAF1, CAP20, MDA-6, SDI1, CDK-interacting protein 1, CDK-interactionprotein 1, cyclin-dependent kinase inhibitor 1, DNA synthesis inhibitor,Melanoma Differentiation Associated Protein, p21CIP, wild typeP53-activated fragment, mDA6, or PCI1, encodes a potent cyclin-dependentkinase inhibitor. The encoded protein binds to and inhibits the activityof cyclin-CDK2 or -CDK4 complexes, and thus functions as a regulator ofcell cycle progression at G1. The expression of this gene is tightlycontrolled by the tumor suppressor protein p53, through which thisprotein mediates the p53-dependent cell cycle G1 phase arrest inresponse to a variety of stress stimuli. This protein can interact withproliferating cell nuclear antigen (PCNA), a DNA polymerase accessoryfactor, and plays a regulatory role in S phase DNA replication and DNAdamage repair. This protein was reported to be specifically cleaved byCASP3-like spases, which thus leads to a dramatic activation of CDK2,and may be instrumental in the execution of apoptosis following caspaseactivation. Multiple alternatively spliced variants have been found forthis gene. DNA and protein sequences of human p21 were previouslyreported, see GenBank Nos. NC_000006.11, NT_007592.15, NC_018917.2 andUniprot No. P38936, each of which is incorporated herein by reference inits entirety for all purposes. Such sequences can be utilized to designprocedures for detection and analysis of the level of p21 activity byways known to one skilled in the art.

Some other components of the CDX2-KLF4 signaling pathway are H3K4demethylase Jarid1b (KDM5B, a.k.a. Plu-1 or Rbp2-h1), microRNA miR-2909,tumor suppressor p53 (a.k.a TP53 or tumor protein (EC:2.7.1.37)),cysteine-aspartic acid protease Caspase-3, Annexin V, B-cellCLL/Lymphoma 2 (BCL2), B-cell CLL/Lymphoma 3 (BCL3), BCL2-associated Xprotein (BAX), bone morphogenetic proteins (BMPs), Wnt (a.k.a. murineint-1), hepatocyte nuclear factor 4α (HNF4α), fibroblast growth factors(Fgf), homeobox (Hox), transcription factor SP1, transcription factorMYC, cyclin D1 (CCND1, a.k.a. PRAD1), and apoptosis-antagonizingtranscription factor (AATF).

An active agent of the present invention can modulate the activity ofone or more component in the CDX2-KLF4 signaling pathway. In someembodiments, the active agent is a small molecule. In some embodiments,the active agent is a polypeptide, such as an antibody. In someembodiments, the active agent is a polynucleotide, such as siRNA.

In some embodiments, the active agent modulates the gene copy number ofa component in the CDX2-KLF4 signaling pathway. In some embodiments, theactive agent can increase or decrease the gene copy number by 0.5×,1.0×, 1.5×, 2×, 3×, 4×, 5×, 6×, 7×, 8×, 9×, 10×, 100×, 1000×, 10000× ormore when compared to the gene copy number before treatment.

In some embodiments, the active agent modulates the mRNA abundance of acomponent in the CDX2-KLF4 signaling pathway. In some embodiments, theactive agent can increase or decrease the mRNA abundance by 0.5×, 1.0×,1.5×, 2×, 3×, 4×, 5×, 6×, 7×, 8×, 9×, 10×, 100×, 1000×, 10000× or morewhen compared to the mRNA abundance before treatment.

In some embodiments, the active agent modulates the protein level of acomponent in the CDX2-KLF4 signaling pathway. In some embodiments, theactive agent can increase or decrease the protein level by 0.5×, 1.0×,1.5×, 2×, 3×, 4×, 5×, 6×, 7×, 8×, 9×, 10×, 100×, 1000×, 10000× or morewhen compared to the protein level before treatment.

In some embodiments, the active agent modulates the mRNA and/or proteinstability of a component in the CDX2-KLF4 signaling pathway. In someembodiments, the active agent can increase or decrease the stabilitywhen compared to the stability before treatment.

In some embodiments, the active agent modulates the enzymatic activityof a component in the CDX2-KLF4 signaling pathway. In some embodiments,the active agent can increase or decrease the enzymatic activity whencompared to the stability before treatment.

The activity of a component in the CDX2-KLF4 signaling pathway can bedetermined by any suitable methods known to one skilled in the art. Insome embodiments, a biological sample is taken from a subject andanalyzed. In some embodiments, the biological sample is then assayed foractivity of a component in the CDX2-KLF4 signaling pathway, such as geneamplification number, RNA, mRNA, cDNA, cRNA, protein, etc.

In some embodiments, mRNA from a biological sample is directly used indetermining the level of activity. In some embodiments, the level isdetermined by hybridization. In some embodiments, the RNA is transformedinto cDNA (complementary DNA) copy using methods known in the art. Insome particular embodiments, the cDNA is labeled with a fluorescentlabel or other detectable label. The cDNA is then hybridized to asubstrate containing a plurality of probes of interest. A probe ofinterest typically hybridizes under stringent hybridization conditionsto at least one DNA sequence of a gene signature. In certainembodiments, the plurality of probes are capable of hybridizing to thesequences derived from the gene biomarkers under the hybridizationconditions. In some embodiments, the conditions comprise using 6×SSC(0.9 M NaCl, 0.09 M sodium citrate, pH 7.4) at 65° C. The probes maycomprise nucleic acids. The term “nucleic acid” encompasses knownnucleotide analogs or modified backbone residues or linkages, which aresynthetic, naturally occurring, and non-naturally occurring, which havesimilar binding properties as the reference nucleic acid, and which aremetabolized in a manner similar to the reference nucleotides. Examplesof such analogs include, without limitation, phosphorothioates,phosphoramidates, methyl phosphonates, chiral-methyl phosphonates,peptide-nucleic acids (PNAs). Methods for detecting can include but arenot limited to RT-PCR, northern blot analyses, gene expression analyses,microarray analyses, gene expression chip analyses, hybridizationtechniques (including FISH), expression beadchip arrays, andchromatography as well as any other techniques known in the art. Methodsfor detecting DNA can include but are not limited to PCR, real-time PCR,digital PCR, hybridization (including FISH), microarray analyses, SNPdetection assays, SNP genotyping assays and chromatography as well asany other techniques known in the art.

In some embodiments, the protein expression level is used in determiningthe level of activity. The protein expression level of a component inthe CDX2-KLF4 signaling pathway can be determined by any suitablemethods known to one skilled in the art. Any suitable methods of proteindetection, quantization and comparison can be used, such as thosedescribed in Tschesche (Methods in Protein Biochemistry, ISBN Walter deGruyter, 2011, ISBN 3110252368, 9783110252361), Goluch et al.(Chip-based detection of protein cancer markers, ProQuest, 2007, ISBN0549463453, 9780549463450), Speicher (Proteome Analysis: Interpretingthe Genome, Elsevier, 2004, ISBN 0080515304, 9780080515304), Albala etal. (Protein Arrays, Biochips and Proteomics, CRC Press, 2003, ISBN0203911121, 9780203911129), Walker (The Protein Protocols Handbook,Springer, 2002, ISBN 0896039404, 9780896039407), Fung (Protein Arrays:Methods and Protocols, Springer, 2004, ISBN 1592597599, 9781592597598),and Bienvenut (Acceleration and Improvement of Protein Identification byMass Spectrometry, Springer, 2005, ISBN 1402033184, 9781402033186), eachof which is incorporated by reference in its entirety for all purposes.In some embodiments, the protein expression level of biomarkers aredetected and measured by immunohistochemistry (IHC), western blot,protein immunostaining, protein immunoprecipitation,immuneeletrophoresis, immunoblotting, BCA assay, spectrophotometry, massspectrometry or enzyme assay, or combinations thereof. For additionalmethods related to detection, quantitation and comparison of biomarkerlevels, see, e.g., Current Protocols in Molecular Biology, Ed. Ausubel,Frederick M. (2010); Current Protocols in Protein Science Last, Ed.Coligan, John E., et al. (2010); Current Protocols in Nucleic AcidChemistry, Ed. Egli, Martin (2010); Current Protocols in Bioinformatics,Ed. Baxevanis, Andreas D. (2010); and Molecular Cloning: A LaboratoryManual, Third Edition, Sambrook, Joseph (2001), all of which areincorporated herein by reference in their entirety.

In some embodiments, antibody of a component in the CDX2-KLF4 signalingpathway can be used as an active agent. In some embodiments, antibody ofa component in the CDX2-KLF4 signaling pathway that negatively controlKLF4 and/or p21, or a component in the CDX2-KLF4 signaling pathway thatare negatively controlled by KLF4 and/or p21 can be used as an activeagent. For example, antibodies of CDX2 can be used to treat the cancersof the present invention.

In some embodiments, antibody of a component in the CDX2-KLF4 signalingpathway can be used to detect the protein level of the component. Insome embodiments, a kit for detection is used. Such antibodies and kitsare available from EMD Millipore, OriGene Custom Assay Services, R&DSystems for biochemical assays, GenScript Custom Assay Services, EnzoLife Sciences for kits & assays, Cloud-Clone Corp. ELISAs, orCloud-Clone Corp. CLIAs. The term “antibody” as used herein is intendedto include monoclonal antibodies, polyclonal antibodies, and chimericantibodies. The antibody may be from recombinant sources and/or producedin transgenic animals. The term “antibody fragment” as used herein isintended to include Fab, Fab′, F(ab′)2, scFv, dsFv, ds-scFv, dimers,minibodies, diabodies, and multimers thereof and biospecific antibodyfragments. Antibodies can be fragmented using conventional techniques.For example, F(ab′)2 fragments can be generated by treating the antibodywith pepsin. The resulting F(ab′)2 fragment can be treated to reducedisulfide bridges to produce Fab′ fragments. Papain digestion can leadto the formation of Fab fragments. Fab, Fab′ and F(ab′)2, scFv, dsFv,ds-scFv, dimers, minibodies, diabodies, biospecific antibody fragmentsand other fragments can also be synthesized by recombinant techniques.

Immunoassays carried out in accordance with the present invention may behomogeneous assays or heterogeneous assays. In a homogeneous assay theimmunological reaction usually involves the specific antibody, a labeledanalyte, and the sample of interest. The signal arising from the labelis modified, directly or indirectly, upon the binding of the antibody tothe labeled analyte. Both the immunological reaction and detection ofthe extent thereof can be carried out in a homogeneous solution.Immunochemical labels which may be employed include free radicals,radioisotopes, fluorescent dyes, enzymes, bacteriophages, or coenzymes.

In a heterogeneous assay approach, the reagents are usually the sample,the antibody, and means for producing a detectable signal. Samples asdescribed above may be used. The antibody can be immobilized on asupport, such as a bead (such as protein A and protein G agarose beads),plate or slide, and contacted with the specimen suspected of containingthe antigen in a liquid phase. The support is then separated from theliquid phase and either the support phase or the liquid phase isexamined for a detectable signal employing means for producing suchsignal. The signal is related to the presence of the analyte in thesample. Means for producing a detectable signal include the use ofradioactive labels, fluorescent labels, or enzyme labels. For example,if the antigen to be detected contains a second binding site, anantibody which binds to that site can be conjugated to a detectablegroup and added to the liquid phase reaction solution before theseparation step. The presence of the detectable group on the solidsupport indicates the presence of the antigen in the test sample.Examples of suitable immunoassays include, but are not limited tooligonucleotides, immunoblotting, immunoprecipitation,immunofluorescence methods, chemiluminescence methods,electrochemiluminescence (ECL) or enzyme-linked immunoassays.

Those skilled in the art will be familiar with numerous specificimmunoassay formats and variations thereof which may be useful forcarrying out the method disclosed herein. See generally E. Maggio,Enzyme-Immunoassay, (1980) (CRC Press, Inc., Boca Raton, Fla.); see alsoU.S. Pat. No. 4,727,022, U.S. Pat. No. 4,659,678, U.S. Pat. No.4,376,110, U.S. Pat. No. 4,275,149, U.S. Pat. No. 4,233,402,U.S. Pat.No. 4,230,767, each of which is herein incorporated by reference in itsentirety for all purposes.

Antibodies can be conjugated to a solid support suitable for adiagnostic assay (e.g., beads such as protein A or protein G agarose,microspheres, plates, slides or wells formed from materials such aslatex or polystyrene) in accordance with known techniques, such aspassive binding. Antibodies as described herein may likewise beconjugated to detectable labels or groups such as radiolabels (e.g.,35S, 125I, 131I), enzyme labels (e.g., horseradish peroxidase, alkalinephosphatase), and fluorescent labels (e.g., fluorescein, Alexa, greenfluorescent protein, rhodamine) in accordance with known techniques.

Antibodies can also be useful for detecting post-translationalmodifications of proteins, polypeptides, mutations, and polymorphisms,such as tyrosine phosphorylation, threonine phosphorylation, serinephosphorylation, glycosylation (e.g., O-GlcNAc). Such antibodiesspecifically detect the phosphorylated amino acids in a protein orproteins of interest, and can be used in immunoblotting,immunofluorescence, and ELISA assays described herein. These antibodiesare well-known to those skilled in the art, and commercially available.Post-translational modifications can also be determined using metastableions in reflector matrix-assisted laser desorption ionization-time offlight mass spectrometry (MALDI-TOF) (Wirth, U. et al. (2002) Proteomics2(10): 1445-51).

In some embodiments, detection reagents can be immobilized on a solidmatrix such as a porous strip to form at least one detection site. Insome embodiments, polynucleotide or polypeptide arrays or microarrayscontaining a plurality of detection agents that hybridize to nucleotideor polypeptide of the biomarkers are utilized. Alternatively, thesubstrate array can be on, e.g., a solid substrate, e.g., a “chip” asdescribed in U.S. Pat. No. 5,744,305. Alternatively, the substrate arraycan be a solution array.

Non-limiting examples for compositions and methods for detecting thecomponents in the CDX2-KLF4 signaling pathway are described in U.S. Pat.Nos. 4,762,706, 5,081,230, 5,300,631, 5,443,956, 7,695,926, 7,785,817,7,479,376, 7,364,868 and U.S. Patent Publication Nos. 20050196793,20110281277, 20120251509, 20050186642, 20140011279, 20110171221,20040235073, 20130011411, and 20130034862, each of which is incorporatedherein by reference in its entirety for all purposes.

Anti-Cancer Compositions

Anti-cancer compositions that can be utilized in the present inventioncomprise at least one active agent. In some embodiments, the activeagent can modulate the activity of the CDX2-KLF4 signaling pathway. Asused herein, the term “modulate” refers to that the compositions canincrease, decrease, eliminate, enhance, delay, reduce, or block theactivity of a component in the CDX2-KLF4 signaling pathway. In someembodiments, the compounds can decrease the CDX2 activity, and/orincrease the KLF4 activity in a human subject. In some embodiments, thecompounds can increase or decrease the activity of one or more upstreamor downstream components in the signaling pathway. In some embodiments,the compounds can increase the activity of one or more downstreamcomponents that are positively regulated by KLF4 (e.g., p21), ordecrease the activity of one or more downstream components in thesignaling pathway that are negatively regulated by KLF4 (e.g., SP1). Insome embodiments, the compounds can decrease the activity of one or moredownstream components that are positively regulated by CDX2, or increasethe activity of one or more downstream components in the signalingpathway that are negatively regulated by CDX2.

Without wishing to be bound by any particular theory, the anti-canceragents of the present invention can act through one or more mechanisms.Such mechanisms include, but are not limited to: (1) inhibition of CDX2activity; (2) induction of KLF4 activity; (3) induction of p21 CDKinhibitor; (4) induction of G1/S Cell cycle arrest; (5) induction ofCaspase 3 enzyme; and (6) induction of apoptosis.

The active agents can be chemical compounds or compositions, biologicalmolecules, or combinations thereof. In some embodiments, the activeagents are small molecules. As used herein, the term “small molecule”refers to a molecule having a molecular weight of less than 500 MW,wherein the drug is a non-peptidyl or peptide agent. In someembodiments, the active agents are antibodies. In some embodiments, theactive agents are antibodies. In some embodiments, the active agents arepolynucleotides, such as siRNA.

In some embodiments, the active agents contain one or more entities thatcan inhibit or decrease the activity of CDX2, e.g., at DNA, RNA, proteinlevel, or combinations thereof.

In some embodiments, the active agents contain one or more antibodiesthat can reduce, inhibit or delay the activity of a component in theCDX2-KLF4 signaling pathway that negatively regulates KLF4, ornegatively regulated by KLF4. In some embodiments, the component can beCDX2, p53, p21, Caspase-3, Annexin V, BAX, BCL2, BCL3, BMP, Wnt, HNF4α,Fgf, Hox, SP1, MYC, CCND1, or AATF. In some embodiments, the activeagents are antibodies. In some embodiments, the active agents are siRNA.For example, the anti-CDX2 agent is an anti-CDX2 antibody. According tothe present invention, an anti-CDX2 antibody at least comprises one ormore anti-CDX2 CDRs.

For example, antisense RNA, ribozyme, dsRNAi, RNA interference (RNAi)technologies can be used in the present invention to target RNAtranscripts of one or more component in the CDX2-KLF4 signaling pathway.Antisense RNA technology involves expressing in, or introducing into, acell an RNA molecule (or RNA derivative) that is complementary to, orantisense to, sequences found in a particular mRNA in a cell. Byassociating with the mRNA, the antisense RNA can inhibit translation ofthe encoded gene product. For example, the anti-CDX2 agents can be smallinterference RNA molecules, such as those disclosed by Wang et al.(“siRNA targeting of Cdx2 inhibits growth of human gastric cancerMGC-803 cells”, World J Gastroenterol. Apr. 28, 2012; 18(16):1903-1914.)

RNA interference (RNAi) is the process of sequence-specific,post-transcriptional gene silencing or transcriptional gene silencing inanimals and plants, initiated by double-stranded RNA (dsRNA) that ishomologous in sequence to the silenced gene. The RNAi technique isdiscussed, for example, in Elibashir, et al., Methods Enzymol. 26:199(2002); McManus & Sharp, Nature Rev. Genetics 3:737 (2002); PCTapplication WO 01/75164; Martinez et al., Cell 110:563 (2002); Elbashiret al., supra; Lagos-Quintana et al., Curr. Biol. 12:735 (2002); Tuschlet al., Nature Biotechnol. 20:446 (2002); Tuschl, Chembiochem. 2:239(2001); Harborth et al., J. Cell Sci. 114:4557 (2001); et al., EMBO J.20:6877 (2001); Lagos-Quintana et al., Science 294:8538 (2001);Hutvagner et al., loc cit, 834; Elbashir et al., Nature 411:494 (2001).

The term “dsRNA” or “dsRNA molecule” or “double-strand RNA effectormolecule” refers to an at least partially double-strand ribonucleic acidmolecule containing a region of at least about 19 or more nucleotidesthat are in a double-strand conformation. The double-stranded RNAeffector molecule may be a duplex double-stranded RNA formed from twoseparate RNA strands or it may be a single RNA strand with regions ofself-complementarity capable of assuming an at least partiallydouble-stranded hairpin conformation (i.e., a hairpin dsRNA or stem-loopdsRNA). In various embodiments, the dsRNA consists entirely ofribonucleotides or consists of a mixture of ribonucleotides anddeoxynucleotides, such as RNA/DNA hybrids. The dsRNA may be a singlemolecule with regions of self-complementarity such that nucleotides inone segment of the molecule base pair with nucleotides in anothersegment of the molecule. In one aspect, the regions ofself-complementarity are linked by a region of at least about 3-4nucleotides, or about 5, 6, 7, 9 to 15 nucleotides or more, which lackscomplementarity to another part of the molecule and thus remainssingle-stranded (i.e., the “loop region”). Such a molecule will assume apartially double-stranded stem-loop structure, optionally, with shortsingle stranded 5′ and/or 3′ ends. In one aspect the regions ofself-complementarity of the hairpin dsRNA or the double-stranded regionof a duplex dsRNA will comprise an Effector Sequence and an EffectorComplement (e.g., linked by a single-stranded loop region in a hairpindsRNA). The Effector Sequence or Effector Strand is that strand of thedouble-stranded region or duplex which is incorporated in or associateswith RISC. In one aspect the double-stranded RNA effector molecule willcomprise an at least 19 contiguous nucleotide effector sequence,preferably 19 to 29, 19 to 27, or 19 to 21 nucleotides, which is areverse complement to the RNA of a target gene, or an opposite strandreplication intermediate, or the anti-genomic plus strand or non-mRNAplus strand sequences of the target gene.

In some embodiments, the dsRNA effector molecule of the invention is a“hairpin dsRNA”, a “dsRNA hairpin”, “short-hairpin RNA” or “shRNA”,i.e., an RNA molecule of less than approximately 400 to 500 nucleotides(nt), or less than 100 to 200 nt, in which at least one stretch of atleast 15 to 100 nucleotides (e.g., 17 to 50 nt, 19 to 29 nt) is basedpaired with a complementary sequence located on the same RNA molecule(single RNA strand), and where said sequence and complementary sequenceare separated by an unpaired region of at least about 4 to 7 nucleotides(or about 9 to about 15 nt, about 15 to about 100 nt, about 100 to about1000 nt) which forms a single-stranded loop above the stem structurecreated by the two regions of base complementarity. The shRNA moleculescomprise at least one stem-loop structure comprising a double-strandedstem region of about 17 to about 100 bp; about 17 to about 50 bp; about40 to about 100 bp; about 18 to about 40 bp; or from about 19 to about29 bp; homologous and complementary to a target sequence to beinhibited; and an unpaired loop region of at least about 4 to 7nucleotides, or about 9 to about 15 nucleotides, about 15 to about 100nt, about 100 to about 1000 nt, which forms a single-stranded loop abovethe stem structure created by the two regions of base complementarity.It will be recognized, however, that it is not strictly necessary toinclude a “loop region” or “loop sequence” because an RNA moleculecomprising a sequence followed immediately by its reverse complementwill tend to assume a stem-loop conformation even when not separated byan irrelevant “stuffer” sequence.

In some embodiments, the active agents contain one or more entities thatcan increase the activity of KLF4, or increase the activity of an entityin the CDX2-KLF4 signaling pathway that is positively regulated by KLF4,or increase the activity of an entity that can positively regulate KLF4,e.g., at DNA, RNA, protein level, or combinations thereof. In someembodiments, the active agent of the present invention is a 2-indolylimidazo[4,5-d]phenanthroline derivative, such as those described in U.S.Pat. No. 8,148,392 or U.S. Patent Publication No. 2007/0123553A1, eachof which is herein incorporated by reference in its entirety for allpurposes.

In some embodiments, the compound has the structure of formula I, or asalt thereof:

Wherein R1 is C1-C4 alkyl; and R2 is halogen. In some embodiments, R¹ ismethyl, isopropyl, or t-butyl.

In some embodiments, the compound has the structure of formula II, or asalt thereof:

In some embodiments, the compound has the structural formula (III), or asalt thereof:

wherein: R1, R2, R3, R4, R6 and R7 are independently selected fromhydrogen, halogen, hydroxyl, thiol, lower alkyl, substituted loweralkyl, lower alkenyl, substituted lower alkenyl, lower alkynyl,substituted lower alkynyl, alkoxy, alkylthio, acyl, aryloxy, amino,amido, carboxyl, aryl, substituted aryl, heterocycle, substitutedheterocycle, heteroalkyl, substituted heteroalkyl, heteroaryl,substituted heteroaryl, cycloalkyl, substituted cycloalkyl, nitro, orcyano or —S(O)1-2R wherein R is alkyl, substituted alkyl, aryl,substituted aryl, heterocycle, heteroaryl, substituted heterocycle, orsubstituted heteroaryl; and wherein R5 is H, alkyl, substituted alkyl,alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, aryl,substituted aryl, heteroaryl, substituted heteroaryl, acyl, —CH2-aryl,—CH2-heteroaryl.

In some embodiments, R1, R2, R3, R4 are independently hydrogen; halogen;C1-C4 alkyl; C1-C4 alkoxy; or C6-C14 aryl; R5 is hydrogen; C1-C4 alkyl;C1-C4 alkyl substituted with C6-C14 aryl; or C4-C6 cycloalkyl; R6 ishydrogen; halogen; C1-C4 alkyl; C1-C4 alkyl substituted with C5-C6heterocycloalkyl wherein the heteroatom is N; C6-C14 aryl; C6-C14 arylsubstituted with C1-C4 alkyl or halo; C5-C6 cycloalkyl; C5-C6heterocycloalkyl; or polycycloalkyl. In some other embodiments, R1, R2,R3, R4 are independently hydrogen; halogen; C1-C4 alkyl; C1-C4 alkoxy;or phenyl; R5 is hydrogen; C1-C4 alkyl; C1-C4 alkyl substituted withphenyl; or cyclopentyl; R6 is hydrogen; halogen; C1-C4 alkyl; C1-C4alkyl substituted with C5-C6 heterocycloalkyl wherein the heteroatom isN; phenyl; phenyl substituted with C1-C4 alkyl or halo; C5-C6cycloalkyl; C5-C6 heterocycloalkyl; or adamantane; and R7 is H.

In some embodiments, said compound has a formula selected from the groupconsisting of

The active agents of the present invention are typically formulatedprior to administration. The present invention thus providespharmaceutical compositions comprising one or more active agents of thepresent invention. In some embodiments, the pharmaceutical compositionscomprise a pharmaceutically acceptable carrier, diluent, or excipient.The pharmaceutical compositions are prepared by known procedures usingwell-known and readily available ingredients.

Active agents of the present invention or pharmaceutical compositionscomprising the active agents may be administered via any suitablemethods, including but not limited to, orally, topically, parenterally,by inhalation or spray, or rectally in dosage unit formulations. In oneembodiment the pharmaceutical composition is administered parenterally,paracancerally, transmucosally, transdermally, intramuscularly,intravenously, intradermally, subcutaneously, intraperitonealy,intraventricularly, intracranially and intratumorally. In someembodiments, the dosage unit formulations contain conventional non-toxicpharmaceutically acceptable carriers, adjuvants and vehicles. In theusual course of therapy, the active agent is incorporated into anacceptable vehicle to form a composition for topical administration tothe affected area, such as hydrophobic or hydrophilic creams or lotions,or into a form suitable for oral, rectal or parenteral administration,such as syrups, elixirs, tablets, troches, lozenges, hard or softcapsules, pills, suppositories, oily or aqueous suspensions, dispersiblepowders or granules, emulsions, injectables, or solutions. The termparenteral as used herein includes, but are not limited to, subcutaneousinjections, intradermal, intra-articular, intravenous, intramuscular,intravascular, intrastemal, intrathecal injection or infusiontechniques.

The present invention also provides for pharmaceutical compositionscomprising one or more of the active agents of the present invention anda vehicle, such as an artificial membrane vesicle (including a liposome,lipid micelle and the like), microparticle or microcapsule.

Compositions intended for oral use may be prepared in either solid orfluid unit dosage forms. Fluid unit dosage form can be preparedaccording to procedures known in the art for the manufacture ofpharmaceutical compositions and such compositions may contain one ormore agents selected from the group consisting of sweetening agents,flavouring agents, colouring agents and preserving agents in order toprovide pharmaceutically elegant and palatable preparations. An elixiris prepared by using a hydroalcoholic (e.g., ethanol) vehicle withsuitable sweeteners such as sugar and saccharin, together with anaromatic flavoring agent. Suspensions can be prepared with an aqueousvehicle with the aid of a suspending agent such as acacia, tragacanth,methylcellulose and the like.

Solid formulations such as tablets contain the active ingredient inadmixture with non-toxic pharmaceutically acceptable excipients that aresuitable for the manufacture of tablets. These excipients may be forexample, inert diluents, such as calcium carbonate, sodium carbonate,lactose, calcium phosphate or sodium phosphate: granulating anddisintegrating agents for example, corn starch, or alginic acid: bindingagents, for example starch, gelatin or acacia, and lubricating agents,for example magnesium stearate, stearic acid or talc and otherconventional ingredients such as dicalcium phosphate, magnesium aluminumsilicate, calcium sulfate, starch, lactose, methylcellulose, andfunctionally similar materials. The tablets may be uncoated or they maybe coated by known techniques to delay disintegration and absorption inthe gastrointestinal tract and thereby provide a sustained action over alonger period. For example, a time delay material such as glycerylmonostearate or glyceryl distearate may be employed.

Formulations for oral use may also be presented as hard gelatin capsuleswherein the active ingredient is mixed with an inert solid diluent, forexample, calcium carbonate, calcium phosphate or kaolin, or as softgelatin capsules wherein the active ingredient is mixed with water or anoil medium, for example peanut oil, liquid paraffin or olive oil. Softgelatin capsules are prepared by machine encapsulation of a slurry ofthe compound with an acceptable vegetable oil, light liquid petrolatumor other inert oil.

Aqueous suspensions contain active materials in admixture withexcipients suitable for the manufacture of aqueous suspensions. Suchexcipients are suspending agents, for example sodiumcarboxylmethylcellulose, methyl cellulose, hydropropylmethylcellulose,sodium alginate, polyvinylpyrrolidone, gum tragacanth and gum acacia:dispersing or wetting agents may be a naturally-occurring phosphatide,for example, lecithin, or condensation products of an alkylene oxidewith fatty acids, for example polyoxyethylene stearate, or condensationproducts of ethylene oxide with long chain aliphatic alcohols, forexample hepta-decaethyleneoxycetanol, or condensation products ofethylene oxide with partial esters derived from fatty acids and ahexitol such as polyoxyethylene sorbitol monooleate, or condensationproducts of ethylene oxide with partial esters derived from fatty acidsand hexitol anhydrides, for example polyethylene sorbitan monooleate.The aqueous suspensions may also contain one or more preservatives, forexample ethyl, or n-propyl-p-hydroxy benzoate, one or more colouringagents, one or more flavouring agents or one or more sweetening agents,such as sucrose or saccharin.

Oily suspensions may be formulated by suspending the active ingredientsin a vegetable oil, for example peanut oil, olive oil, sesame oil orcoconut oil, or in a mineral oil such as liquid paraffin. The oilysuspensions may contain a thickening agent, for example beeswax, hardparaffin or cetyl alcohol. Sweetening agents such as those set forthabove, and flavouring agents may be added to provide palatable oralpreparations. These compositions may be preserved by the addition of ananti-oxidant such as ascorbic acid.

Dispersible powders and granules suitable for preparation of an aqueoussuspension by the addition of water provide the active ingredient inadmixture with a dispersing or wetting agent, suspending agent and oneor more preservatives. Suitable dispersing or wetting agents andsuspending agents are exemplified by those already mentioned above.Additional excipients, for example sweetening, flavouring and colouringagents, may also be present.

Pharmaceutical compositions of the invention may also be in the form ofoil-in-water emulsions. The oil phase may be a vegetable oil, forexample olive oil or peanut oil, or a mineral oil, for example liquidparaffin or mixtures of these. Suitable emulsifying agents may benaturally-occurring gums, for example gum acacia or gum tragacanth,naturally-occurring phosphatides, for example soy bean, lecithin, andesters or partial esters derived from fatty acids and hexitol,anhydrides, for example sorbitan monooleate, and condensation productsof the said partial esters with ethylene oxide, for examplepolyoxyethylene sorbitan monooleate. The emulsions may also containsweetening and flavoring agents.

The pharmaceutical compositions may be in the form of a sterileinjectable aqueous or oleaginous suspension. This suspension may beformulated according to known art using those suitable dispersing orwetting agents and suspending agents that have been mentioned above. Thesterile injectable preparation may also be a sterile injectable solutionor a suspension in a non-toxic parentally acceptable diluent or solvent,for example as a solution in 1,3-butanediol. Among the acceptablevehicles and solvents that may be employed are water, Ringer's solutionand isotonic sodium chloride solution. In addition, sterile, fixed oilsare conventionally employed as a solvent or suspending medium. For thispurpose any bland fixed oil may be employed including synthetic mono- ordiglycerides. In addition, fatty acids such as oleic acid find use inthe preparation of injectables. Adjuvants such as local anesthetics,preservatives and buffering agents can also be included in theinjectable solution or suspension.

In some embodiments, the delivery systems suitable include time-release,delayed release, sustained release, or controlled release deliverysystems. In some embodiments, a composition of the present invention canbe delivered in a controlled release system, such as sustained-releasematrices. Non-limiting examples of sustained-release matrices includepolyesters, hydrogels (e.g., poly(2-hydroxyethyl-methacrylate) asdescribed by Langer et al., 1981, J. Biomed. Mater. Res., 15:167-277 andLanger, 1982, Chem. Tech., 12:98-105), or poly(vinylalcohol)],polylactides (U.S. Pat. No. 3,773,919; EP 58,481), copolymers ofL-glutamic acid and gamma ethyl-L-glutamate (Sidman et al., 1983,Biopolymers, 22:547-556), non-degradable ethylene-vinyl acetate (Langeret al., supra), degradable lactic acid-glycolic acid copolymers such asthe LUPRON DEPOT™ (injectable microspheres composed of lacticacid-glycolic acid copolymer and leuprolide acetate), andpoly-D-(−)-3-hydroxybutyric acid (EP 133,988). In some embodiments, thecomposition may be administered using intravenous infusion, animplantable osmotic pump, a transdermal patch, liposomes, or other modesof administration. In one embodiment, a pump may be used (see Langer,supra; Sefton, CRC Crit. Ref. Biomed. Eng. 14:201 (1987); Buchwald etal., Surgery 88:507 (1980); Saudek et al., N. Engl. J. Med. 321:574(1989). In another embodiment, polymeric materials can be used. In yetanother embodiment, a controlled release system can be placed inproximity to the therapeutic target, for example liver, thus requiringonly a fraction of the systemic dose (see, e.g., Goodson, in MedicalApplications of Controlled Release, supra, vol. 2, pp. 115-138 (1984).Other controlled release systems are discussed in the review by Langer(Science 249:1527-1533 (1990).

In some embodiments, the release of the composition occurs in bursts.Examples of systems in which release occurs in bursts includes, e.g.,systems in which the composition is entrapped in liposomes which areencapsulated in a polymer matrix, the liposomes being sensitive tospecific stimuli, e.g., temperature, pH, light or a degrading enzyme andsystems in which the composition is encapsulated by an ionically-coatedmicrocapsule with a microcapsule core degrading enzyme.

In some embodiments, the release of the composition isgradual/continuous. Examples of systems in which release of theinhibitor is gradual and continuous include, e.g., erosional systems inwhich the composition is contained in a form within a matrix andeffusional systems in which the composition penneates at a controlledrate, e.g., through a polymer. Such sustained release systems can bee.g., in the form of pellets, or capsules.

Other embodiments of the compositions administered according to theinvention incorporate particulate forms, protective coatings, proteaseinhibitors or permeation enhancers for various routes of administration,such as parenteral, pulmonary, nasal and oral.

The pharmaceutical compositions of the present invention may beadministered, together or separately, in the form of suppositories forrectal administration of the drug. These compositions can be prepared bymixing the drug with a suitable non-irritating excipient which is solidat ordinary temperatures but liquid at the rectal temperature and willtherefore melt in the rectum to release the drug. Such materials includecocoa butter and polyethylene glycols.

Other pharmaceutical compositions and methods of preparingpharmaceutical compositions are known in the art and are described, forexample, in “Remington: The Science and Practice of Pharmacy” (formerly“Remingtons Pharmaceutical Sciences”); Gennaro, A., Lippincott, Williams& Wilkins, Philadelphia, Pa. (2000).

The pharmaceutical compositions of the present invention may beadministered to a subject by a variety of routes depending on the cancerto be treated, for example, the compositions may be administered orally,topically, parenterally, by inhalation or spray, or rectally in dosageunit formulations. In one embodiment, the compounds are administeredsystemically to a subject, for example, by bolus injection or infusioninto a subject's bloodstream or by oral administration. When used inconjunction with one or more known chemotherapeutic agents, thecompounds can be administered prior to, or after, administration of thechemotherapeutic agents, or they can be administered concomitantly. Theone or more chemotherapeutic may also be administered systemically, forexample, by bolus injection, infusion, or oral administration.

The pharmaceutical compositions of the present invention may be used aspart of a neo-adjuvant therapy (to primary therapy), or as part of anadjuvant therapy regimen. The present invention contemplates the use ofthe pharmaceutical compositions of the present invention at variousstages in tumor development and progression, including, but not limitedto, in the treatment of advanced and/or aggressive neoplasias (i.e.overt disease in a subject that is not amenable to cure by localmodalities of treatment, such as surgery or radiotherapy), metastaticdisease, locally advanced disease and/or refractory tumors (i.e. acancer or tumor that has not responded to treatment). As used herein,the term “primary therapy” refers to a first line of treatment upon theinitial diagnosis of cancer in a subject. Exemplary primary therapiesmay involve surgery, a wide range of chemotherapies and radiotherapy.“Adjuvant therapy” refers to a therapy that follows a primary therapyand that is administered to subjects at risk of relapsing. Adjuvantsystemic therapy is usually begun soon after primary therapy to delayrecurrence, prolong survival or cure a subject.

The pharmaceutical compositions of the present invention can be usedalone or in combination with one or more other anti-cancer agents, suchas chemotherapeutic agents as part of a primary therapy or an adjuvanttherapy. Combinations of the pharmaceutical compositions of the presentinvention and standard chemotherapeutics may act to improve the efficacyof the chemotherapeutic and, therefore, can be used to improve standardcancer therapies. This application can be important in the treatment ofdrug-resistant cancers which are not responsive to standard treatment.Drug-resistant cancers can arise, for example, from heterogeneity oftumor cell populations, alterations in response to chemotherapy andincreased malignant potential. Such changes are often more pronounced atadvanced stages of disease.

The pharmaceutical compositions of the present invention can be usedalone or in combination with radiation therapeutic. In some embodiments,the radiation therapeutic is administered at a dosage of about 40 Gy toabout 80 Gy. In some embodiments the dosage is about 50 Gy to about 70Gy, in some embodiments, the dosage is about 50 Gy to about 65 Gy. Insome embodiments, the radiation therapy is administered at a dosage ofabout 50 Gy, about 55 Gy, about 60 Gy or about 65 Gy.

The dosage to be administered is not subject to defined limits, but itwill usually be an effective amount. It will usually be the equivalent,on a molar basis of the pharmacologically active free form produced froma dosage formulation upon the metabolic release of the active free drugto achieve its desired pharmacological and physiological effects. Thecompositions may be formulated in a unit dosage form. The term “unitdosage form” refers to physically discrete units suitable as unitarydosages for human subjects and other mammals, each unit containing apredetermined quantity of active material calculated to produce thedesired therapeutic effect, in association with a suitablepharmaceutical excipient.

In some embodiments, the active agents are compounds having thestructure of formulas described herein. However, it will be understoodthat the actual amount of the compound(s) to be administered will bedetermined by a physician, in the light of the relevant circumstances,including the condition to be treated, the chosen route ofadministration, the actual compound administered, the age, weight, andresponse of the individual patient, and the severity of the patient'ssymptoms. The above dosage range is given by way of example only and isnot intended to limit the scope of the invention in any way. In someinstances dosage levels below the lower limit of the aforesaid range maybe more than adequate, while in other cases still larger doses may beemployed without causing harmful side effects, for example, by firstdividing the larger dose into several smaller doses for administrationthroughout the day.

Dosages for a particular individual can be determined by one of ordinaryskill in the art using conventional considerations, (e.g. by means of anappropriate, conventional pharmacological protocol). A physician may,for example, prescribe a relatively low dose at first, subsequentlyincreasing the dose until an appropriate response is obtained. The doseadministered to an individual is sufficient to effect a beneficialtherapeutic response in the individual over time, or, e.g., to reducesymptoms, or other appropriate activity, depending on the application.The dose is determined by the efficacy of the particular formulation,and the activity, stability or serum half-life of the composition andthe condition of the individual, as well as the body weight or surfacearea of the individual to be treated. The size of the dose is alsodetermined by the existence, nature, and extent of any adverseside-effects that accompany the administration of a particular vector,formulation, or the like in a particular individual.

In some embodiments, dosage unit for a compound of the present inventioncan be about 0.001 mg, about 0.002 mg, about 0.003 mg, about 0.004 mg,about 0.005 mg, about 0.006 mg, about 0.007 mg, about 0.008 mg, about0.009 mg, about 0.01 mg, about 0.02 mg, about 0.03 mg, about 0.04 mg,about 0.05 mg, about 0.06 mg, about 0.07 mg, about 0.08 mg, about 0.09mg, about 0.1 mg, about 0.2 mg, about 0.3 mg, about 0.4 mg, about 0.5mg, about 0.6 mg, about 0.7 mg, about 0.8 mg, about 0.9 mg, about 1 mg,about 2 mg, about 3 mg, about 4 mg, about 5 mg, about 6 mg, about 7 mg,about 8 mg, about 9 mg, about 10 mg, about 15 mg, about 20 mg, about 30mg, about 35 mg, about 40 mg, about 45 mg, about 50 mg, about 60 mg,about 70 mg, about 80 mg, about 90 mg, about 100 mg, about 200 mg, about300 mg, about 400 mg, about 500 mg or more.

In some embodiments, daily dosages of the compounds of the presentinvention will typically be about 0.001 mg/kg of body weight, about0.002 mg/kg of body weight, about 0.003 mg/kg of body weight, about0.004 mg/kg of body weight, about 0.005 mg/kg of body weight, about0.006 mg/kg of body weight, about 0.007 mg/kg of body weight, about0.008 mg/kg of body weight, about 0.009 mg/kg of body weight, about 0.01mg/kg of body weight, about 0.02 mg/kg of body weight, about 0.03 mg/kgof body weight, about 0.04 mg/kg of body weight, about 0.05 mg/kg ofbody weight, about 0.06 mg/kg of body weight, about 0.07 mg/kg of bodyweight, about 0.08 mg/kg of body weight, about 0.09 mg/kg of bodyweight, about 0.1 mg/kg of body weight, about 0.2 mg/kg of body weight,about 0.3 mg/kg of body weight, about 0.4 mg/kg of body weight, about0.5 mg/kg of body weight, about 0.6 mg/kg of body weight, about 0.7mg/kg of body weight, about 0.8 mg/kg of body weight, about 0.9 mg/kg ofbody weight, about 1 mg/kg of body weight, about 2 mg/kg of body weight,about 3 mg/kg of body weight, about 4 mg/kg of body weight, about 5mg/kg of body weight, about 6 mg/kg of body weight, about 7 mg/kg ofbody weight, about 8 mg/kg of body weight, about 9 mg/kg of body weight,about 10 mg/kg of body weight, about 15 mg/kg of body weight, about 20mg/kg of body weight, about 30 mg/kg of body weight, about 35 mg/kg ofbody weight, about 40 mg/kg of body weight, about 45 mg/kg of bodyweight, about 50 mg/kg of body weight, about 60 mg/kg of body weight,about 70 mg/kg of body weight, about 80 mg/kg of body weight, about 90mg/kg of body weight, about 100 mg/kg of body weight, about 200 mg/kg ofbody weight, about 300 mg/kg of body weight, about 400 mg/kg of bodyweight, about 500 mg/kg of body weight or more. Either single or divideddose can be used.

In some embodiments, the dosage form of the present invention iseffective to provide a maximum total plasma concentration in said humansubject of no more than about 1 pg/mL, about 5 pg/mL, about 10 pg/mL,about 50 pg/mL, about 100 pg/mL, about 500 pg/mL, about 1 ng/mL, about 5ng/mL, about 10 ng/mL, about 50 ng/mL, about 100 ng/mL, about 500 ng/mL,about 1 μg/mL, about 5 μg/mL, about 10 μg/mL, about 50 μg/mL, about 100μg/mL, about 500 μg/mL, about 1 mg/mL or more of free base equivalent ofthe compound.

In a further aspect of any of dosage forms as described herein, thedosage form is an oral dosage form. In a still further aspect, thedosage form is an intravenous dosage form. The dosage form is suitablefor administration to a human subject particularly for use in thetreatment of any of the disorders described herein. In some embodiments,the dosage form is a subcutaneous dosage form.

Pharmaceutical compositions comprising one or more compounds describedherein in combination with one or more other anti-cancer agents can alsobe used. Such conjoint treatment may be achieved by way of thesimultaneous, sequential or separate dosing of the individual componentsof the treatment.

In some embodiments, the anti-cancer agents are chemotherapeutics. Insome embodiments, the chemotherapeutics are selected from alkylatingagents, anti-metabolites, anti-microtubule agents, Topoisomeraseinhibitors, and Cytotoxic antibiotics. Examples of chemotherapeuticagents include, but are not limited to, paclitaxel (Taxol®), docetaxel(Taxotere®), cisplatin, carboplatin (Paraplatin®), gemcitabinehydrochloride (Gemzar®), doxorubicin, etoposide (Etopophos®, Vepesid®),pemetrexed (Alimta®), topotecan (Hycamtin®), vinblastine (Velbe®),Vindesine (Eldisine®), vinorelbine (Navelbine®), ifosfamide (Mitoxana®),Mitomycin, and gemcitabine. These agents may be given in combination,for example, vinorelbine and cisplatin or carboplatin; gemcitabine withcisplatin or carboplatin or paclitaxel; MIC (mitomycin, ifosfamide andcisplatin); MVP (mitomycin, vinblastine and cisplatin); and EC(etoposide and carboplatin). Examples of useful chemotherapeutic drugsinclude, but are not limited to, hydroxyurea, busulphan, cisplatin,carboplatin, chlorambucil, melphalan, cyclophosphamide, Ifosphamide,danorubicin, doxorubicin, epirubicin, mitoxantrone, vincristine,vinblastine, Navelbine® (vinorelbine), etoposide, teniposide,paclitaxel, docetaxel, gemcitabine, cytosine, arabinoside, bleomycin,neocarcinostatin, suramin, taxol, mitomycin C and the like.

As used herein, the term “alkylating agents” refers to agents that havethe ability to alkylate molecules in a subject, including proteins, RNAand DNA. Non-limiting examples of alkylating agents include nitrogenmustards, nitrosoureas, tetrazines, aziridines, cisplatins andderivatives, and non-classical alkylating agents. Nitrogen mustardsinclude mechlorethamine, cyclophosphamide, melphalan, chlorambucil,ifosfamide and busulfan. Nitrosoureas include N-Nitroso-N-methylurea(MNU), carmustine (BCNU), lomustine (CCNU) and semustine (MeCCNU),fotemustine and streptozotocin. Tetrazines include dacarbazine,mitozolomide and temozolomide. Aziridines include thiotepa, mytomycinand diaziquone (AZQ). Cisplatin and derivatives include cisplatin,carboplatin and oxaliplatin. They impair cell function by formingcovalent bonds with the amino, carboxyl, sulfhydryl, and phosphategroups in biologically important molecules. [20] Non-classicalalkylating agents include procarbazine and hexamethylmelamine.

As used herein, the term “anti-metabolites” refers to molecule thatimpedes DNA, RNA, or protein synthesis. In some embodiments,anti-metabolites resemble either nucleobases or nucleosides (anucleotide without the phosphate group), but have altered chemicalgroups. These drugs exert their effect by either blocking the enzymesrequired for DNA synthesis or becoming incorporated into DNA or RNA. Byinhibiting the enzymes involved in DNA synthesis, they prevent mitosisbecause the DNA cannot duplicate itself. Also, after misincorporation ofthe molecules into DNA, DNA damage can occur and programmed cell death(apoptosis) is induced. In some embodiments, the anti-metabolites areanti-folates, fluoropyrimidines, deoxynucleoside analogues andthiopurines. In some embodiments, the anti-metabolites are selected frommethotrexate, pemetrexed, fluorouracil, capecitabine, cytarabine,gemcitabine, decitabine, Vidaza, fludarabine, nelarabine, cladribine,clofarabine, pentostatin, thioguanine and mercaptopurine.

As used herein, the term “anti-microtubule agents” refers to chemicalsthat block cell division by preventing microtubule function.Representative examples of such agents include taxanes (e.g., paclitaxel(discussed in more detail below) and docetaxel) (Schiff et al., Nature277: 665-667, 1979; Long and Fairchild, Cancer Research 54: 4355-4361,1994; Ringel and Horwitz, J. Natl. Cancer Inst. 83(4): 288-291, 1991;Pazdur et al, Cancer Treat. Rev. 19(4): 351-386, 1993), campothecin,mitoxantrone, eleutherobin (e.g., U.S. Pat. No. 5,473,057),sarcodictyins (including sarcodictyin A), epothilones A and B (Bollag etal., Cancer Research 55: 2325-2333, 1995), discodermolide (ter Haar etal., Biochemistry 35: 243-250, 1996), deuterium oxide (D20) (James andLefebvre, Genetics 130(2): 305-314, 1992; Sollott et al., J. Clin.Invest. 95: 1869-1876, 1995), hexylene glycol (2-methyl-2,4-pentanediol)(Oka et al., Cell Struct. Funct. 16(2): 125-134, 1991), tubercidin(7-deazaadenosine) (Mooberry et al., Cancer Lett. 96(2): 261-266, 1995),LY290181 (2-amino-4-(3-pyridyl)-4H-naphtho(1,2-b)pyran-3-cardonitrile)(Panda et al., J. Biol. Chem. 272(12): 7681-7687, 1997; Wood et al.,Mol. Pharmacol. 52(3): 437-444, 1997), aluminum fluoride (Song et al.,J. Cell. Sci. Suppl. 14: 147-150, 1991), ethylene glycolbis-(succinimidylsuccinate) (Caplow and Shanks, J. Biol. Chem. 265(15):8935-8941, 1990), glycine ethyl ester (Mejillano et al., Biochemistry31(13): 3478-3483, 1992), nocodazole (Ding et al., J. Exp. Med 171(3):715-727, 1990; Dotti et al., J. Cell Sci. Suppl. 15: 75-84, 1991; Oka etal., Cell Struct. Funct. 16(2): 125-134, 1991; Weimer et al., J. Cell.Biol. 136(1), 71-80, 1997), cytochalasin B (Illinger et al., Biol. Cell73(2-3): 131-138, 1991), colchicine and CI 980 (Allen et al., Am. J.Physiol. 261(4 Pt. 1): L315-L321, 1991; Ding et al., J. Exp. Med.171(3): 715-727, 1990; Gonzalez et al., Exp. Cell. Res. 192(1): 10-15,1991; Stargell et al., Mol. Cell. Biol. 12(4): 1443-1450, 1992; Garciaet al., Antican. Drugs 6(4): 533-544, 1995), colcemid (Barlow et al.,Cell. Motil. Cytoskeleton 19(1): 9-17, 1991; Meschini et al., J.Microsc. 176(Pt. 3): 204-210, 1994; Oka et al., Cell Struct. Funct.16(2): 125-134, 1991), podophyllotoxin (Ding et al., J. Exp. Med.171(3): 715-727, 1990), benomyl (Hardwick et al., J. Cell. Biol. 131(3):709-720, 1995; Shero et al., Genes Dev. 5(4): 549-560, 1991), oryzalin(Stargell et al., Mol. Cell. Biol. 12(4): 1443-1450, 1992),majusculamide C (Moore, J. Ind. Microbiol. 16(2): 134-143, 1996),demecolcine (Van Dolah and Ramsdell, J. Cell. Physiol. 166(1): 49-56,1996; Wiemer et al., J. Cell. Biol. 136(1): 71-80, 1997),methyl-2-benzimidazolecarbamate (MBC) (Brown et al., J. Cell. Biol.123(2): 387-403, 1993), LY195448 (Barlow & Cabral, Cell Motil. Cytoskel.19: 9-17, 1991), subtilisin (Saoudi et al., J. Cell Sci. 108: 357-367,1995), 1069C85 (Raynaud et al., Cancer Chemother Pharmacol. 35: 169-173,1994), steganacin (Hamel, Med. Res. Rev. 16(2): 207-231, 1996),combretastatins (Hamel, Med Res. Rev. 16(2): 207-231, 1996), curacins(Hamel, Med. Res. Rev. 16(2): 207-231, 1996), estradiol (Aizu-Yokata etal., Carcinogen. 15(9): 1875-1879, 1994), 2-methoxyestradiol (Hamel,Med. Res. Rev. 16(2): 207-231, 1996), flavanols (Hamel, Med. Res. Rev.16(2): 207-231, 1996), rotenone (Hamel, Med Res. Rev. 16(2): 207-231,1996), griseofulvin (Hamel, Med Res. Rev. 16(2): 207-231, 1996), vincaalkaloids, including vinblastine and vincristine (Ding et al., J. Exp.Med 171(3): 715-727, 1990; Dirk et al., Neurochem. Res. 15(11):1135-1139, 1990; Hamel, Med. Res. Rev. 16(2): 207-231, 1996; Illinger etal., Biol. Cell 73(2-3): 131-138, 1991; Wiemer et al., J. Cell. Biol.136(1): 71-80, 1997), maytansinoids and ansamitocins (Hamel, Med Res.Rev. 16(2): 207-231, 1996), rhizoxin (Hamel, Med. Res. Rev. 16(2):207-231, 1996), phomopsin A (Hamel, Med. Res. Rev. 16(2): 207-231,1996), ustiloxins (Hamel, Med. Res. Rev. 16(2): 207-231, 1996),dolastatin 10 (Hamel, Med Res. Rev. 16(2): 207-231, 1996), dolastatin 15(Hamel, Med. Res. Rev. 16(2): 207-231, 1996), halichondrins andhalistatins (Hamel, Med. Res. Rev. 16(2): 207-231, 1996), spongistatins(Hamel, Med. Res. Rev. 16(2): 207-231, 1996), cryptophycins (Hamel, Med.Res. Rev. 16(2): 207-231, 1996), rhazinilam (Hamel, Med. Res. Rev.16(2): 207-231, 1996), betaine (Hashimoto et al., Zool. Sci. 1: 195-204,1984), taurine (Hashimoto et al., Zool. Sci. 1: 195-204, 1984),isethionate (Hashimoto et al., Zool. Sci. 1: 195-204, 1984), HO-221(Ando et al., Cancer Chemother Pharmacol. 37: 63-69, 1995),adociasulfate-2 (Sakowicz et al., Science 280: 292-295, 1998),estramustine (Panda et al., Proc. Natl. Acad. Sci. USA 94: 10560-10564,1997), monoclonal anti-idiotypic antibodies (Leu et al., Proc. Natl.Acad. Sci. USA 91(22): 10690-10694, 1994), microtubule assemblypromoting protein (taxol-like protein, TALP) (Hwang et al., Biochem.Biophys. Res. Commun. 208(3): 1174-1180, 1995), cell swelling induced byhypotonic (190 mosmol/L) conditions, insulin (100 mmol/L) or glutamine(10 mmol/L) (Haussinger et al., Biochem. Cell. Biol. 72(1-2): 12-19,1994), dynein binding (Ohba et al., Biochim. Biophys. Acta 1158(3):323-332, 1993), gibberelin (Mita and Shibaoka, Protoplasma 119(1/2):100-109, 1984), XCHOI (kinesin-like protein) (Yonetani et al., Mol.Biol. Cell 7(suppl): 211 A, 1996), lysophosphatidic acid (Cook et al.,Mol. Biol. Cell 6(suppl): 260A, 1995), lithium ion (Bhattacharyya andWolff, Biochem. Biophys. Res. Commun. 73(2): 383-390, 1976), plant cellwall components (e.g., poly-L-lysine and extensin) (Akashi et al.,Planta 182(3): 363-369, 1990), glycerol buffers (Schilstra et al.,Biochem. J. 277(Pt. 3): 839-847, 1991; Farrell and Keates, Biochem.Cell. Biol. 68(11): 1256-1261, 1990; Lopez et al., J. Cell. Biochem.43(3): 281-291, 1990), Triton X-100 microtubule stabilizing buffer(Brown et al., J. Cell Sci. 104(Pt. 2): 339-352, 1993; Safiejko-Mroczkaand Bell, J. Histochem. Cytochem. 44(6): 641-656, 1996), microtubuleassociated proteins (e.g., MAP2, MAP4, tau, big tau, ensconsin,elongation factor-1-alpha (EF-1α) and E-MAP-115) (Burgess et al., CellMotil. Cytoskeleton 20(4): 289-300, 1991; Saoudi et al., J. Cell. Sci.108(Pt. 1): 357-367, 1995; Bulinski and Bossler, J. Cell. Sci. 107(Pt.10): 2839-2849, 1994; Ookata et al., J. Cell Biol. 128(5): 849-862,1995; Boyne et al., J. Comp. Neurol 358(2): 279-293, 1995; Ferreira andCaceres, J. Neurosci. 11(2): 392-400, 1991; Thurston et al., Chromosoma105(1):20-30, 1996; Wang et al., Brain Res. Mol. Brain Res. 38(2):200-208, 1996; Moore and Cyr, Mol. Biol. Cell 7(suppl): 221-A, 1996;Masson and Kreis, J. Cell Biol. 123(2), 357-371, 1993), cellularentities (e.g., histone H1, myelin basic protein and kinetochores)(Saoudi et al., J. Cell. Sci. 108(Pt. 1): 357-367, 1995; Simerly et al.,J. Cell Biol. 111(4): 1491-1504, 1990), endogenous microtubularstructures (e.g., axonemal structures, plugs and GTP caps) (Dye et al.,Cell Motil. Cytoskeleton 21(3): 171-186, 1992; Azhar and Murphy, CellMotil. Cytoskeleton 15(3): 156-161, 1990; Walker et al., J. Cell Biol.114(1): 73-81, 1991; Drechsel and Kirschner, Curr. Biol. 4(12):1053-1061, 1994), stable tubule only polypeptide (e.g., STOP145 andSTOP220) (Pirollet et al., Biochim. Biophys. Acta 1160(1): 113-119,1992; Pirollet et al., Biochemistry 31(37): 8849-8855, 1992; Bosc etal., Proc. Natl. Acad. Sci. USA 93(5): 2125-2130, 1996; Margolis et al.,EMBO J. 9(12): 4095-4102, 1990) and tension from mitotic forces (Nicklasand Ward, J. Cell Biol. 126(5): 1241-1253, 1994), as well as anyanalogues and derivatives of any of the above. Such compounds can act byeither depolymerizing microtubules (e.g., colchicine and vinblastine),or by stabilizing microtubule formation (e.g., paclitaxel).

In some embodiments, an anti-tumor agent include mitotic inhibitors, forexample vinca alkaloid derivatives such as vinblastine vinorelbine,vindescine and vincristine; colchines allochochine, halichondrine,N-benzoyltrimethyl-methyl ether colchicinic acid, dolastatin 10,maystansine, rhizoxine, taxanes such as taxol (paclitaxel), docetaxel(Taxotere), 2′-N-[3-(dimethylamino)propyl]glutaramate (taxolderivative), thiocholchicine, trityl cysteine, teniposide, methotrexate,azathioprine, fluorouricil, cytocine arabinoside,2′2′-difluorodeoxycytidine (gemcitabine), adriamycin and mitamycin.Alkylating agents, for example cis-platin, carboplatin oxiplatin,iproplatin, Ethyl ester of N-acetyl-DL-sarcosyl-L-leucine (Asaley orAsalex), 1,4-cyclohexadiene-1,4-dicarbamic acid,2,5-bis(1-azirdinyl)-3,6-dioxo-, diethyl ester (diaziquone),1,4-bis(methanesulfonyloxy)butane (bisulfan or leucosulfan)chlorozotocin, clomesone, cyanomorpholinodoxorubicin, cyclodisone,dianhydroglactitol, fluorodopan, hepsulfam, mitomycin C,hycantheonemitomycin C, mitozolamide,1-(2-chloroethyl)-4-(3-chloropropyl)-piperazine dihydrochloride,piperazinedione, pipobroman, porfiromycin, spirohydantoin mustard,teroxirone, tetraplatin, thiotepa, triethylenemelamine, uracil nitrogenmustard, bis(3-mesyloxypropyl)amine hydrochloride, mitomycin,nitrosoureas agents such as cyclohexyl-chloroethylnitrosourea,methylcyclohexyl-chloroethylnitrosourea1-(2-chloroethyl)-3-(2,6-dioxo-3-piperidyl)-1-nitroso-urea,bis(2-chloroethyl)nitrosourea, procarbazine, dacarbazine, nitrogenmustard-related compounds such as mechloroethamine, cyclophosphamide,ifosamide, melphalan, chlorambucil, estramustine sodium phosphate,strptozoin, and temozolamide. DNA anti-metabolites, for example5-fluorouracil, cytosine arabinoside, hydroxyurea,2-[(3hydroxy-2-pyrinodinyl)methylene]-hydrazinecarbothioamide,deoxyfluorouridine, 5-hydroxy-2-formylpyridine thiosemicarbazone,alpha-2′-deoxy-6-thioguanosine, aphidicolin glycinate,5-azadeoxycytidine, beta-thioguanine deoxyriboside, cyclocytidine,guanazole, inosine glycodialdehyde, macbecin II, pyrazolimidazole,cladribine, pentostatin, thioguanine, mercaptopurine, bleomycin,2-chlorodeoxyadenosine, inhibitors of thymidylate synthase such asraltitrexed and pemetrexed disodium, clofarabine, floxuridine andfludarabine. DNA/RNA antimetabolites, for example, L-alanosine,5-azacytidine, acivicin, aminopterin and derivatives thereof such asN-[2-chloro-5-[[(2,4-diamino-5-methyl-6-quinazolinyl)methyl]amino]benzoyl]-L-asparticacid,N-[4-[[(2,4-diamino-5-ethyl-6-quinazolinyl)methyl]amino]benzoyl]-L-asparticacid,N-[2-chloro-4-[[(2,4-diaminopteridinyl)methyl]amino]benzoyl]-L-asparticacid, soluble Baker's antifol, dichloroallyl lawsone, brequinar, ftoraf,dihydro-5-azacytidine, methotrexate, N-(phosphonoacetyl)-L-aspartic acidtetrasodium salt, pyrazofuran, trimetrexate, plicamycin, actinomycin D,cryptophycin, and analogs such as cryptophycin-52 or, for example, oneof the preferred anti-metabolites disclosed in European PatentApplication No. 239362 such asN-(5-[N-(3,4-dihydro-2-methyl-4-oxoquinazolin-6-ylmethyl)-N-methylamino]-2-thenoyl)-L-glutamicacid; growth factor inhibitors; cell cycle inhibitors; intercalatingantibiotics, for example adriamycin and bleomycin; proteins, for exampleinterferon; and anti-hormones, for example anti-estrogens such asNolvadex™ (tamoxifen) or, for example anti-androgens such as Casodex™(4′-cyano-3-(4-fluorophenylsulphonyl)-2-hydroxy-2-methyl-3′-(trifluoromethyl)propionanilide).

As used herein, the term “topoisomerase inhibitors” refers to agentsthat can modulate the activity of topoisomerase I and/or topoisomeraseII. In some embodiments, the topoisomerase inhibitor of this inventioncan be a topoisomerase I inhibitor, which can be, in some embodiments, acamptothecin derivative. A camptothecin derivative of this invention canbe, but is not limited to Belotecan (CKD602), Camptothecin,7-Ethyl-10-Hydroxy-CPT, 10-Hydroxy-CPT, Rubitecan (9-Nitro-CPT),7-Ethyl-CPT, Topotecan, Irinotecan, Silatecan (DB67) and anycombinations thereof. In some embodiments of this invention, thetopoisomerase I inhibitor can be an indenoisoquinoline derivative, whichcan be but is not limited to NSC706744, NSC725776, NSC724998 and anycombinations thereof. In further embodiments of this invention, thetopoisomerase inhibitor is a topoisomerase II inhibitor, which in someembodiments can be an acridine derivative, which can be but is notlimited to Amsacrine, in some embodiments the topoisomerase II inhibitorcan be a podophyllotoxin derivative, which can be but is not limited toetoposide, and in some embodiments the topoisomerase II inhibitor can bea bisdioxopiperazine derivative, which can be but is not limited toICRF-193, dexrazoxane (ICRF-187) and any combinations thereof. In yetfurther embodiments of this invention, the topoisomerase inhibitor canbe Resveratrol (PMID: 20304553; PMID: 15796584)41, Epigallocatechingallate (PMID: 18293940; PMID: 11594758; PMID: 11558576; PMID:1313232)42,43, Genistein (PMID: 17458941)44, Daidzein (PMID:17458941)45. Quercetin (PMID: 1313232; PMID: 16950806; PMID: 15312049),natural flavones related to quercetin that inhibit topoisomerase, suchas acacetin, apigenin, kaempferol and morin (PMID: 8567688)46-48,Luteolin (PMID: 12027807; PMID: 16950806; PMID: 15312049)46; Myricetin(PMID: 20025993)49 and any combinations thereof. In certain embodiments,the topoisomerase inhibitor can be an interfering RNA (RNAi) moleculethat targets topoisomerase I, topoisomerase II or both. Nonlimitingexamples of RNAi molecules include small interfering RNA (siRNA), shorthairpin RNA (shRNA), microRNA (miRNA), antisense nucleic acid molecules,and the like as are well known in the art. Nonlimiting examples ofsiRNAs and shRNAs of this invention are provided in Table 2. In someembodiments, a zinc finger nuclease, an antibody and/or a ribozyme canbe employed to inhibit topoisomerase activity in the methods of thisinvention.

As used herein, the term “cytotoxic antibiotics” cytotoxic antibioticsinclude, but are not limited to, antinomycin, bleomycin, mitomycin,plicamycin and the like. Examples of tyrosine kinase inhibitors include,but are not limited to, nilotinib, imatinib, gefitinib, erlotinib,cetuximab, panitumumab, zalutumumab, nimotuzumab, matuzuman and thelike.

In some embodiments, the other anti-cancer agents are monoclonalantibodies, such as alemtuzumab, bevacizumab, cetuximab, gemtuzumab,rituximab, and trastuzumab; photosensitizers, such as aminolevulinicacid, methyl aminolevulinate, porfimer sodium, and verteporfin; andother agents, such as alitretinoin, altretamine, amsacrine, anagrelide,arsenic trioxide, asparaginase, bexarotene, bortezomib, celecoxib,denileukin diftitox, erlotinib, estramustine, gefitinib,hydroxycarbamide, imatinib, pentostatin, masoprocol, mitotane,pegaspargase, and tretinoin.

In some embodiments, the other anti-cancer agents are those can be usedto treat leukemia, such as drugs for Acute Lymphoblastic Leukemia (ALL),Acute Myeloid Leukemia (AML), Chronic Lymphocytic Leukemia (CLL),Chronic Myelogenous Leukemia (CML), and Meningeal Leukemia, include butare not limited to, Abitrexate (Methotrexate), Adriamycin PFS(Doxorubicin Hydrochloride), Adriamycin RDF (Doxorubicin Hydrochloride),Arranon (Nelarabine), Asparaginase Erwinia chrysanthemi, Cerubidine(Daunorubicin Hydrochloride), Clafen (Cyclophosphamide), Clofarabine,Clofarex (Clofarabine), Clolar (Clofarabine), Cyclophosphamide,Cytarabine, Cytosar-U (Cytarabine), Cytoxan (Cyclophosphamide),Dasatinib, Daunorubicin Hydrochloride, Doxorubicin Hydrochloride,Erwinaze (Asparaginase Erwinia Chrysanthemi), Folex (Methotrexate),Folex PFS (Methotrexate), Gleevec (Imatinib Mesylate), Iclusig(Ponatinib Hydrochloride), Imatinib Mesylate, Marqibo (VincristineSulfate Liposome), Mercaptopurine, Methotrexate, Methotrexate LPF(Methorexate), Mexate (Methotrexate), Mexate-AQ (Methotrexate),Nelarabine, Neosar (Cyclophosphamide), Oncaspar (Pegaspargase),Pegaspargase, Purinethol (Mercaptopurine), Rubidomycin (DaunorubicinHydrochloride), Sprycel (Dasatinib), Tarabine PFS (Cytarabine), VincasarPFS, incristine Sulfate), Vincristine Sulfate, Vincristine SulfateLiposome, Hyper-CVAD, Adriamycin PFS (Doxorubicin Hydrochloride),Adriamycin RDF (Doxorubicin Hydrochloride), Arsenic Trioxide, Cerubidine(Daunorubicin Hydrochloride), Clafen (Cyclophosphamide),Cyclophosphamide, Cytarabine, Cytosar-U (Cytarabine), Cytoxan(Cyclophosphamide), Daunorubicin Hydrochloride, DoxorubicinHydrochloride, Neosar (Cyclophosphamide), Rubidomycin (DaunorubicinHydrochloride), Tarabine PFS (Cytarabine), Trisenox (Arsenic Trioxide),Vincasar PFS (Vincristine Sulfate), Vincristine Sulfate, ADE,Alemtuzumab, Ambochlorin (Chlorambucil), Amboclorin (Chlorambucil),Arzerra (Ofatumumab), Bendamustine, Hydrochloride, Campath(Alemtuzumab), Chlorambucil, Clafen (Cyclophosphamide),Cyclophosphamide, Cytoxan (Cyclophosphamide), Fludara (FludarabinePhosphate), Fludarabine Phosphate, Gazyva (Obinutuzumab), Ibrutinib,Imbruvica (Ibrutinib), Leukeran (Chlorambucil), Linfolizin(Chlorambucil), Neosar (Cyclophosphamide), Obinutuzumab, Ofatumumab,Treanda (Bendamustine Hydrochloride), CHLORAMBUCIL-PREDNISONE, CVP,Bosulif (Bosutinib), Bosutinib, Busulfan, Busulfex (Busulfan), Clafen(Cyclophosphamide), Cyclophosphamide, Cytarabine, Cytosar-U(Cytarabine), Cytoxan (Cyclophosphamide), Dasatinib, Gleevec (ImatinibMesylate), Iclusig (Ponatinib Hydrochloride), Imatinib Mesylate, Myleran(Busulfan), Neosar (Cyclophosphamide), Nilotinib, OmacetaxineMepesuccinate, Ponatinib Hydrochloride, Sprycel (Dasatinib), Synribo(Omacetaxine Mepesuccinate), Tarabine PFS (Cytarabine), Tasigna(Nilotinib), Cytarabine, Cytosar-U (Cytarabine), and Tarabine PFS(Cytarabine).

In some embodiments, additional anti-cancer agents that can be usedtogether with the compounds of the present invention include agents thatcan inhibit EGFR (epidermal growth factor receptor) responses, such asEGFR antibodies, EGF antibodies, and molecules that are EGFR inhibitors;VEGF (vascular endothelial growth factor) inhibitors; and erbB2 receptorinhibitors, such as organic molecules or antibodies that bind to theerbB2 receptor. EGFR inhibitors are described in, for example in WO95/19970, WO 98/14451, WO 98/02434 and U.S. Pat. No. 5,747,498.EGFR-inhibiting agents include, but are not limited to, the monoclonalantibodies C225 and anti-EGFR 22Mab (ImClone Systems Incorporated of NewYork, N.Y., USA), the compounds ZD-1839 (AstraZeneca), BIBX-1382(Boehringer Ingelheim), MDX-447 (Medarex Inc. of Annandale, N.J., USA),and OLX-103 (Merck & Co. of Whitehouse Station, N.J., USA), VRCTC-310(Ventech Research) and EGF fusion toxin (Seragen Inc. of Hopkinton,Mass.). VEGF inhibitors, for example AG-13736 (Pfizer, Inc.), can alsobe combined or co-administered with the composition. VEGF inhibitors aredescribed in, for example in WO 99/24440, WO 95/21613, WO 99/61422, U.S.Pat. No. 5,834,504, WO 98/50356 (published Nov. 12, 1998), U.S. Pat. No.5,883,113, U.S. Pat. No. 5,886,020, U.S. Pat. No. 5,792,783, U.S. Pat.No. 6,534,524, WO 99/10349, WO 97/32856, WO 97/22596, WO 98/54093, andWO 98/02437, all of which are herein incorporated by reference in theirentirety. Other examples of some specific VEGF inhibitors are IM862(Cytran Inc. of Kirkland, Wash., USA); Avastin™ or bevacizumab, ananti-VEGF monoclonal antibody (Genentech, Inc. of South San Francisco,Calif.); and angiozyme, a synthetic ribozyme from Ribozyme (Boulder,Colo.) and Chiron (Emeryville, Calif.). ErbB2 receptor inhibitors, suchas GW-282974 (Glaxo Wellcome plc), and the monoclonal antibodies AR-209(Aronex Pharmaceuticals Inc. of The Woodlands, Tex., USA) and 2B-1(Chiron), may be administered in combination with the composition. SucherbB2 inhibitors include those described in WO 98/02434, WO 99/35146, WO99/35132, WO 98/02437, WO 97/13760, WO 95/19970, U.S. Pat. No.5,587,458, and U.S. Pat. No. 5,877,305, each of which is hereinincorporated by reference in its entirety.

In some embodiments, a continuous dosing schedule is used for thetreatment. In some embodiments, a compound of a dosage form comprisingthe compound of the present invention is administered once per day,twice per day, three times per day, or more times per day, orcontinuously during a period of a day, or continuously through out aday, on a daily schedule, or every other day, every third day, everyforth day, every fifth day, every sixth day, or every week. Thetreatment can last a period of time determined by doctors, or until thecancer cells are completely or significantly inhibited.

In some embodiments, an intermittent dosing schedule is used for thetreatment. Such treatment is especially useful if there is toxicityconcern. In some embodiments,

The following examples illustrate various aspects of the invention. Theexamples should, of course, be understood to be merely illustrative ofonly certain embodiments of the invention and not to constitutelimitations upon the scope of the invention.

EXAMPLES Example 1 In Vitro Antiproliferative Activity of LOR-253 inLeukemia, Lymphoma and Multiple Myeloma Cell Lines

Antiproliferative activity of LOR-253 was determined by XTT assay asfollows. Cells (4×10³/well) in 100 μL of growth medium were seeded in96-well cell culture plates and incubated overnight at 37° C. The mediumwas removed and replaced with a total volume of 100 μL growth mediumcontaining LOR-253 (or 0.1% DMSO vehicle control), as described in therespective experiments. Suspension cells were plated (4-6×10³/well) in50 μL growth medium, and 50 μL growth medium containing LOR-253 (or 0.1%DMSO vehicle control) was added to each well. After incubation of thecells at 37° C. for 5 days, cell viability was quantitated with the useof sodium3′-[1-(phenylamino-carbonyl)-3,4-tetrazolium}-bis(4-methoxy-6-nitro)benzenesulfonic acid hydrate (XTT) colorimetric assay (Roche). XTT labelingreagent (1 mg/mL) was mixed with electron-coupling reagent, followingthe manufacturer's instructions, and 50 μL of the mixture was addeddirectly to the cells. The plates were further incubated at 37° C. for 4h, and the absorbance of each well was measured at 490 nm with amultiwell spectrophotometer (Bio-Tek Instruments Inc.). The data wereadjusted relative to the blank and expressed as a percentage of cellgrowth compared with the vehicle control. (Ref. Huesca et al., MolCancer Ther. 2009. 8:2586-96; Lorus publication on LOR-133)

The in vitro essay results (FIG. 3) indicate that leukemia/lymphoma,including AML cell lines, such as HL-60, MV 411, THP-1, HEL92.1.7,CCRF-CEM, MOLT-4, Jurkat, K-562, Ramos, and Raji, are the cell linesmost sensitive to LOR-253, while other cell lines of lung cancer,bladder cancer, colon cancer, prostate cancer, melanoma, and breastcancer, are less sensitive to LOR-253. The variability in sensitivity toLOR-253 may be attributable to the extent of innate KLF4 suppression.The IC₅₀ values of LOR-253 for each leukemia and lymphoma cell lines areshown below.

Cancer Type Cell Line IC₅₀ (μM) Acute myeloid leukemia Kasumi-1 0.0069KG-1 0.033 HL-60 0.046 EOL-1 0.058 NB4 0.06 MV-4-11 0.069 OCI-AML2 0.076THP-1 0.077 MOLM13 (CD47+) 0.13 NOMO-1 0.121 SKM-1 0.145 HEL92.1.7 0.305Acute lymphoblastic leukemia CCRF-CEM 0.039 MOLT-4 0.07 Jurkat 0.071Chronic Myelogenous Leukemia K562 0.25 Non-Hodgkin's Lymphoma Ramos0.125 Raji 0.011 CA46 0.19 Toledo 0.077 DB 0.099 Mino 0.14 RL 0.19Multiple myeloma MM.1R 0.15 U266B1 0.18 HuNS1 0.072

Example 2 KLF4/p21 Induction in AML Cell Lines In Vitro

To determine if KLF4 and/or p21 expressions are induced by LOR-253, AMLcells (THP1, HL-60) were treated with DMSO (vehicle control) or 0.5 μMLOR-253 for 16 hours. Total RNA was extracted using the TRIzol Plus RNAPurification kit (Ambion, Life Technologies), according to themanufacturer's instructions. First strand cDNA was synthesized from 1-2ug total RNA using random hexamer primers (Invitrogen) and theSuperScript II Reverse Transcriptase kit (Invitrogen). QuantitativeRT-PCR was performed in the ABI Prism 7000 Sequence Detection Systemusing cDNAs and human TaqMan Gene Expression Assay primer/probe sets forKruppel-like factor 4 (KLF4), cyclin-dependent kinase inhibitor 1A (p21)and the ABI TaqMan Universal PCR master mix protocol. Gene expressionwas normalized with β-actin gene expression in the same sample, and foldchanges in KLF4 or p21 were expressed relative to the correspondingexpression level in the DMSO treated samples using the comparative CTmethod. Treatment of THP1 and HL-60 AML cell lines with LOR-253 resultsin increased expression KLF4 and p21 (See FIG. 4).

Example 3 Treatment with LOR-253 Results in G1/S Cell Cycle Arrest inAML Cell Lines

THP1 and HL-60 AML cell lines were treated with DMSO (vehicle control)or 0.5 μM LOR-253 for 16 hours. Cells were washed once in PBS+1% FCS andfixed using ice cold 70% ethanol overnight. Fixed cells were washedtwice, resuspended in PI/RNaseA solution, containing 20 μg/mL propidiumiodide and 250 μg/mL RNaseA, and stained for 30 minuets at 37° C.Stained cells were analyzed using a BD FACSCalibur flow cytometer. Theresults indicate treatment with LOR-253 results in G1/S cell cyclearrest in AML cell lines (FIG. 5)

Example 4 Treatment with LOR-253 Induces Apoptosis in Cell Lines

THP-1 cells were treated with DMSO, 0.5 or 1 uM of LOR-253 for 16 hours.Cells were washed twice with cold PBS and resuspended Annexin V bindingbuffer. 1×10⁵ cells in 100 μL were stained with FITC-Annexin V andpropidium iodide, and incubated for 15 minutes at room temperature.After staining, 400 ul of binding buffer was added and the cells werekept on ice until analyzed on a BD FACSCanto flow cytometer. THP-1 cellstreated with LOR-253 showed elevated Annexin V staining, indicatinginduction of apoptosis. Increasing concentration of LOR-253 used totreat the cells resulted in an increase in the early apoptoticpopulation (Q3: Annexin V+/PI−). The results indicate treatment withLOR-253 induces apoptosis in AML cell lines (FIG. 6).

THP1 and HL-60 cells were treated with DMSO (vehicle control) or 0.5 μMLOR-253 for 48 hours. Cell lysates were collected using a 1% Triton-X100lysis. Caspase 3 activity was measured using EnzChek Caspase-3 Assay Kit#1 (Life Technologies) with 5 μg of cell lysates according tomanufacturer's protocol. Treatment of THP1 and HL-60 cells with LOR-253results in elevated Caspase 3 activity, indicating induction ofapoptosis (FIG. 7).

THP1 cells were treated with DMSO (vehicle control) or 0.5 μM LOR-253for 48 hours. Total RNA was extracted using the TRIzol Plus RNAPurification kit (Ambion, Life Technologies), according to themanufacturer's instructions. First strand cDNA was synthesized from 1-2ug total RNA using random hexamer primers (Invitrogen) and theSuperScript II Reverse Transcriptase kit (Invitrogen). QuantitativeRT-PCR was performed in the ABI Prism 7000 Sequence Detection Systemusing cDNAs and human TaqMan Gene Expression Assay primer/probe sets forBCL2-associated X protein (BAX), B-cell CLL/Lymphoma 2 (BCL2) and theABI TaqMan Universal PCR master mix protocol. Gene expression wasnormalized with β-actin gene expression in the same sample, and foldchanges in BAX or BCL2 were expressed relative to the correspondingexpression level in the DMSO treated samples using the comparative CTmethod. Elevation of BAX and repression of BCL2 upon treatment of THP1cells with LOR-253 indicates induction of apoptosis (FIG. 8).

Example 5 In Vivo Efficacy of LOR-253 HCL in H226 Xenograft Model—Doseand Schedule

H226 model mice were treated according to several administrationschedules as shown in the table below.

Administration Schedule/cycle Treatment Group Dose level (days) (1 cycle= 28 days) 1 n = 8 10 mg/kg-iv 2T-12B- 2T 2 cycles 2 n = 8 10 mg/kg-iv3T-12B-3T 2 cycles 3 n = 8 10 mg/kg-iv 2T-5B-2T 2 cycles 4 n = 8 10mg/kg-iv 3T-5B-3T 2 cycles 5 n = 8 Vehicle alone 3T-5B-3T 2 cycles T =consecutive days treatment B = break for one

The results show that groups 2, 3, and 4 are effectively treated byLOR-253 (FIG. 9). The results indicates that schedule with one week gapis superior to two-week gap, and suggests weekly administration scheduleis preferred.

Example 6 In Vivo Dose-Dependent Pharmacokinetic (PK) andPharmacodynamic (PD) Responses in Xenograft CD-1 Nude Mice Treated byLOR-253 HCL

To study the pharmacokinetic (PK) in tumor cells treated by LOR-253,CD-1 nude mice were treated by i.v. bolus injections of LOR-253 HCl at1, 5, and 15 mg/kg. The serum level of LOR-253 was measured. The resultindicates that the serum level of LOR-253 has a dose related increase(FIG. 10).

To study the pharmacodynamic (PD) responses, mice were treated for 5consecutive days with 1, 5, and 15 mg/kg LOR-253. Tumors were measured16 hours after the last dose, and the KLF-4 protein level was measured.Average KLF4 protein levels increased in a dose-dependent manner,correlating with tumor biodistribution and dose-response antitumoractivity (FIG. 11).

Example 7 LOR-253 for Treating AML, Phase I Clinical Trial

Preclinical testing established a broad therapeutic index for LOR-253:efficacy of LOR-253 was demonstrated in xenograft models againstmultiple tumor types; extensive GLP safety and PK studies were performedin rats and dogs; no treatment-related cardiovascular effects wereobserved in repeat-dose toxicity study of dogs at highest dose tested(data not shown); no significant inhibition of hERG tail current densityor CYP450 enzymes; and GLP blood compatibility studies confirmed thesuitability of IV formulation.

Phase I: This was an open-label, phase 1 study to determine the maximumtolerated dose (MTD) or appropriate target dose if MTD not reached toidentify the recommended phase 2 dose of LOR-253 HCl in patients withadvanced or metastatic solid tumours. In the first set of doses, LOR-253HCl was given in ascending doses until the maximum administered dose orappropriate target dose is reached. In the other set of doses, LOR-253HCl was given in ascending doses starting from 20 mg/m2 until themaximum administered dose or appropriate target dose is reached. Patientas treated on LOR-253 HCl for 2 cycles for the evaluation.

-   Other Name: No other names are used.-   Study Type: Interventional-   Study Design:    -   Endpoint Classification: Safety Study    -   Intervention Model: Single Group Assignment    -   Masking: Open Label    -   Primary Purpose: Treatment-   Official Title: Open-Label, Phase 1 Study of LOR-253 HCl in Patients    With Advanced or Metastatic Solid Tumours    Primary Outcome Measures:    -   To determine the maximum tolerated dose (MTD) or appropriate        target dose if MTD not reached to identify the recommended phase        2 dose of LOR-253 HCl in patients with advanced or metastatic        solid tumours. [Time Frame: 8 weeks]        Secondary Outcome Measures:    -   To characterize the safety profile of LOR-253 HCl when        administered to patients with advanced or metastatic solid        tumours. [Time Frame: 8 weeks]        Inclusion Criteria:    -   1. Male or female 18 years of age or older.    -   2. Histologically confirmed diagnosis of solid tumor for which        no effective therapy is available or that is unresponsive to        conventional therapy.    -   3. Meet laboratory parameter requirements at study entry.        Exclusion Criteria:    -   1. Chemotherapy, radiotherapy, biologic therapy, immunotherapy        or any other investigational drugs within 21 days of beginning        study treatment with LOR-253 HCl.    -   2. A hematologic malignancy.    -   3. A history of brain or other central nervous system        metastases.    -   4. Have a presence of a significant infection.    -   5. Clinically significant autoimmune disease.    -   6. Uncontrolled intercurrent illness.    -   7. With iron or copper overload syndromes.    -   8. Pregnancy or breast feeding.

Safety and Antitumor Activity Demonstrated in Phase I Trial:

-   -   Dose-Escalating Study in Patients with Advanced Solid Tumors    -   Excellent Safety Profile (N=27 Patients) across 7 dose levels    -   Most common AE and SAE: Fatigue and 1 case of reversible        hypophosphatemia    -   Stable Disease achieved in 41% of evaluable patients; No RECIST        PR    -   Tumor shrinkage (FIG. 12) in patient with NSCLC (poorly        differentiated adenocarcinoma) and extensive metastases        refractory to prior standard and investigational therapies

The phase I trial results demonstrated that LOR-253 is a safe and activedrug for treating solid tumors.

Example 8 In Vitro Antiproliferative Activity of LOR-253 inMyelodysplastic Syndromes (MDS), Acute Lymphocytic Leukemia (ALL),Chronic Myelogenous Leukemia (CML), Adult T-Cell Leukaemia (ATLL),Lymphoma, Gastric Cancer, and Multiple Myeloma Cell Lines

Antiproliferative activity of LOR-253 in MDS, ALL, CML, ATLL, lymphoma,gastric cancer, and multiple myeloma cell lines are determined by XTTassay as described in Example 1.

MDS cell lines tested in the assay include, but are not limited to,TER-3 (Mishima et al., New human myelodysplastic cell line, TER-3: G-CSFspecific downregulation of Ca2+/calmodulin-dependent protein kinase IV,J Cell Physiol. 2002 May; 191(2):183-90), MDS92 (Tohyama et al., A novelfactor-dependent human myelodysplastic cell line, MDS92, containshaemopoietic cells of several lineages, Br J Haematol. 1995 December;91(4):795-9), and SKM-1 (Kimura et al., Antiproliferative and AntitumorEffects of Azacitidine Against the Human Myelodysplastic Syndrome CellLine SKM-1, Anticancer Research 32:795-798, 2002), each of which isherein incorporated by reference in its entirety.

ALL cell lines tested in the assay include, but are not limited to,CCRF-CEM (Foley et al., Continuous culture of human lymphoblasts fromperipheral blood of a child with acute leukemia. Cancer 196518:522-529), MOLT-4 (Minowada et al., Rosette-forming human lymphoidcell lines. I. Establishment and evidence for origin of thymus-derivedlymphocytes. Journal of the National Cancer Institute 1972 49 (3):891-895), and Jurkat (Schneider et al., Characterization of EBV-genomenegative “null” and “T” cell lines derived from children with acutelymphoblastic leukemia and leukemic transformed non-Hodgkin lymphoma.Int J Cancer. 1977 May 15; 19(5):621-6).

CML cell lines tested in the assay include, but are not limited to, K562(Drexler, Leukemia cell lines: in vitro models for the study of chronicmyeloid leukemia. Leuk Res. 1994 December; 18(12):919-27).

Lymphoma cell lines tested in the assay include, but are not limited to,Ramos, Raji, CA46, Toledo, DB, Mino, and RL.

Gastric cancer cell lines tested in the assay include, but are notlimited to, AGS (ATCC Accession No. CRL-1739TM), SNU-1 (ATCC AccessionNo. CRL-5971TM), SNU-5 (ATCC Accession No. CRL-5973TM), SNU-16 (ATCCAccession No. CRL-5974TM), Hs 746T (ATCC Accession No. HTB-135TM),NCI-N87 (N87; ATCC Accession No. CRL-5822TM), KATO III (ATCC AccessionNo. HTB-103TM), SNU-520, SNU-719, NUGC-4, STKM-2, MKN-45, MKN-74, 20M,AKG, ECC4, G42LATE, GCIY, GCIY, GT3TKB, H-111, H-162, H-30, H-55,HGC-27, HSC-39, HUG-1N, JR1, KWS, MKN-1, MKN-28, MKN-7, MKN-74, MKN-74,MKN-74, NCI-N87, NUGC-3, OKAJIMA, SK-GT-1, SK-GT-2, SK-GT-5, SNU-16,SNU-216, SNU-484, SNU-55, SNU-601, SNU-638, SNU-668, TGBC11TKB,TGBC11TKB, TMK-1, and YCC-3

Multiple myeloma cell lines tested in the assay include, but are notlimited to, HMCLs described in Lombardi et al. (Molecularcharacterization of human multiple myeloma cell lines by integrativegenomics: insights into the biology of the disease. Genes ChromosomesCancer. 2007 March; 46(3):226-38.), XGs, NANs, BCN, MDN, SBN HMCLs,U266, RPMI8226, RPMI1640, ANBL-6, KMS-11, KMS12-BM, KMS12-PE, KMM1, LP1,L363, OPM2, NCIH929, JIM3, MO-1, LP1, L363, NCIH929, OPM2, UHKT-89,SKMM2, U266B1 (Nilsson et al., Established immunoglobulin producingmyeloma (IgE) and lymphoblastoid (IgG) cell lines from an IgE myelomapatient. Clin Exp Immunol. 1970 October; 7(4):477-89), MM.1R (Greensteinet al., Characterization of the MM.1 human multiple myeloma (MM) celllines: a model system to elucidate the characteristics, behavior, andsignaling of steroid-sensitive and -resistant MM cells. Exp Hematol.2003 April; 31(4):271-82), and HuNS1 (Winkelhake, Myelomas for producinghuman/human hybridomas. U.S. Pat. No. 4,720,459 dated Jan. 19, 1988).

The in vitro essay results indicate that one or more cell lines of MDS,ALL, CML, ATLL, lymphoma, gastric cancer, or multiple myeloma cell linesare sensitive to LOR-253.

Example 9 KLF4/p21 Induction in Myelodysplastic Syndromes (MDS), ALL,CML, ATLL, Lymphoma, Gastric Cancer, and Multiple Myeloma Cell Lines InVitro

To determine if KLF4 and/or p21 expressions are induced by LOR-253,myelodysplastic syndromes (MDS), ALL, CML, ATLL, lymphoma, gastriccancer, and multiple myeloma cell lines described in Example 8 aretreated with DMSO (vehicle control) or 0.1, 0.2, 0.5 or 1 μM LOR-253 for16 hours. Total RNA is extracted using the TRIzol Plus RNA Purificationkit (Ambion, Life Technologies), according to the manufacturer'sinstructions. First strand cDNA is synthesized from 1-2 ug total RNAusing random hexamer primers (Invitrogen) and the SuperScript II ReverseTranscriptase kit (Invitrogen). Quantitative RT-PCR is performed in theABI Prism 7000 Sequence Detection System using cDNAs and human TaqManGene Expression Assay primer/probe sets for Krüppel-like factor 4(KLF4), cyclin-dependent kinase inhibitor 1A (p21) and the ABI TaqManUniversal PCR master mix protocol. Gene expression is normalized withβ-actin gene expression in the same sample, and fold changes in KLF4 orp21 are expressed relative to the corresponding expression level in theDMSO treated samples using the comparative CT method. Treatment of oneor more myelodysplastic syndromes (MDS), ALL, CML, ATLL, lymphoma,gastric cancer, or multiple myeloma cell lines with LOR-253 results inincreased expression KLF4 and/or p21.

Example 10 Treatment with LOR-253 Results in G1/S Cell Cycle Arrest inMyelodysplastic Syndromes (MDS), ALL, CML, ATLL, Lymphoma, GastricCancer, and Multiple Myeloma Cell Lines

Myelodysplastic syndromes (MDS), ALL, CML, ATLL, lymphoma, gastriccancer, and multiple myeloma cell lines described in Example 8 aretreated with DMSO (vehicle control) or 0.1, 0.2, 0.5 or 1 μM LOR-253 for16 hours. Cells are washed once in PBS+1% FCS and fixed using ice cold70% ethanol overnight. Fixed cells are washed twice, resuspended inPI/RNaseA solution, containing 20 μg/mL propidium iodide and 250 μg/mLRNaseA, and stained for 30 minuets at 37° C. Stained cells are analyzedusing a BD FACSCalibur flow cytometer. The results indicate treatmentwith LOR-253 results in G1/S cell cycle arrest in one or moremyelodysplastic syndromes (MDS), ALL, CML, ATLL, lymphoma, gastriccancer, or multiple myeloma cell lines.

Example 11 Treatment with LOR-253 Induces Apoptosis in MyelodysplasticSyndromes (MDS), ALL, CML, ATLL, Lymphoma, Gastric Cancer, and MultipleMyeloma Cell Lines

Myelodysplastic syndromes (MDS), ALL, CML, ATLL, lymphoma, gastriccancer, and multiple myeloma cell lines described in Example 8 aretreated with DMSO, 0.1, 0.2, 0.5 or 1 μM of LOR-253 for 16 hours. Cellsare washed twice with cold PBS and resuspended Annexin V binding buffer.1×10⁵ cells in 100 μL are stained with FITC-Annexin V and propidiumiodide, and incubated for 15 minutes at room temperature. Afterstaining, 400 ul of binding buffer is added and the cells are kept onice until analyzed on a BD FACSCanto flow cytometer. One or more celllines treated with LOR-253 show elevated Annexin V staining, indicatinginduction of apoptosis. Increasing concentration of LOR-253 used totreat the cells results in an increase in the early apoptotic population(Q3: Annexin V+/PI−).

Cell lines are also treated with DMSO (vehicle control) or 0.1, 0.2, 0.5or 1 μM LOR-253 for 48 hours. Cell lysates are collected using a 1%Triton-X100 lysis. Caspase 3 activity is measured using EnzChekCaspase-3 Assay Kit #1 (Life Technologies) with 5 μg of cell lysatesaccording to manufacturer's protocol. Treatment of one or more celllines with LOR-253 results in elevated Caspase 3 activity, indicatinginduction of apoptosis.

Total RNA of treated cell lines are extracted using the TRIzol Plus RNAPurification kit (Ambion, Life Technologies), according to themanufacturer's instructions. First strand cDNA is synthesized from 1-2μg total RNA using random hexamer primers (Invitrogen) and theSuperScript II Reverse Transcriptase kit (Invitrogen). QuantitativeRT-PCR is performed in the ABI Prism 7000 Sequence Detection Systemusing cDNAs and human TaqMan Gene Expression Assay primer/probe sets forBCL2-associated X protein (BAX), B-cell CLL/Lymphoma 2 (BCL2) and theABI TaqMan Universal PCR master mix protocol. Gene expression isnormalized with β-actin gene expression in the same sample, and foldchanges in BAX or BCL2 are expressed relative to the correspondingexpression level in the DMSO treated samples using the comparative CTmethod. Elevation of BAX and repression of BCL2 upon treatment of one ormore myelodysplastic syndromes (MDS), ALL, CML, ATLL, lymphoma, gastriccancer, or multiple myeloma cell lines with LOR-253 indicates inductionof apoptosis.

Example 12 In Vivo Efficacy of LOR-253 HCL in Xenograft Model ofMyelodysplastic Syndromes (MDS), ALL, CML, ATLL, Lymphoma, GastricCancer, and Multiple Myeloma

Model mice of myelodysplastic syndromes (MDS), ALL, CML, ATLL, lymphoma,gastric cancer, and multiple myeloma are created according to standardprocedures. The model mice are treated according to severaladministration schedules as shown in the table below.

Treatment Administration (1 cycle = 7, 14, 21, Group Dose levelSchedule/cycle or 28 days) 1 n = 8 1 mg/kg-iv 2T-12B- 2T 1, 2, 3, or 4cycles 2 n = 8 1 mg/kg-iv 3T-12B-3T 1, 2, 3, or 4 cycles 3 n = 8 1mg/kg-iv 2T-5B-2T 1, 2, 3, or 4 cycles 4 n = 8 1 mg/kg-iv 3T-5B-3T 1, 2,3, or 4 cycles 5 n = 8 5 mg/kg-iv 2T-12B- 2T 1, 2, 3, or 4 cycles 6 n =8 5 mg/kg-iv 3T-12B-3T 1, 2, 3, or 4 cycles 7 n = 8 5 mg/kg-iv 2T-5B-2T1, 2, 3, or 4 cycles 8 n = 8 5 mg/kg-iv 3T-5B-3T 1, 2, 3, or 4 cycles 9n = 8 10 mg/kg-iv 2T-12B- 2T 1, 2, 3, or 4 cycles 10 n = 8 10 mg/kg-iv3T-12B-3T 1, 2, 3, or 4 cycles 11 n = 8 10 mg/kg-iv 2T-5B-2T 1, 2, 3, or4 cycles 12 n = 8 10 mg/kg-iv 3T-5B-3T 1, 2, 3, or 4 cycles 13 n = 8 20mg/kg-iv 2T-12B- 2T 1, 2, 3, or 4 cycles 14 n = 8 20 mg/kg-iv 3T-12B-3T1, 2, 3, or 4 cycles 15 n = 8 20 mg/kg-iv 2T-5B-2T 1, 2, 3, or 4 cycles16 n = 8 20 mg/kg-iv 3T-5B-3T 1, 2, 3, or 4 cycles 17 n = 8 Vehiclealone 3T-5B-3T 1, 2, 3, or 4 cycles T = consecutive days treatment B =break for one

The results show that one or more groups are effectively treated byLOR-253.

Example 13 In Vivo Dose-Dependent Pharmacokinetic (PK) andPharmacodynamic (PD) Responses in Xenograft Model Mice Treated byLOR-253 HCL with Myelodysplastic Syndromes (MDS), ALL, CML, ATLL,Lymphoma, Gastric Cancer, and Multiple Myeloma

To study the pharmacokinetic (PK) in myelodysplastic syndromes (MDS),ALL, CML, ATLL, lymphoma, gastric cancer, and multiple myeloma tumorcells treated by LOR-253, CD-1 nude mice are treated by i.v. bolusinjections of LOR-253 HCl at 1, 5, and 15 mg/kg. The serum level ofLOR-253 in each treatment is measured.

To study the pharmacodynamic (PD) responses, mice are treated for 5consecutive days with 1, 5, and 15 mg/kg LOR-253. Tumors are measured 16hours after the last dose, and the KLF-4 protein level is measured. Thisresults show that average KLF4 protein levels in one or more treatmentincrease in a dose-dependent manner, correlating with tumorbiodistribution and dose-response antitumor activity.

Example 14 In Vivo Efficacy of LOR-253 HCL in a Kasumi-1 AML XenograftModel

The anti-tumor activity of LOR-253 HCl was evaluated in another in vivoanimal model of human AML. The human AML cell line Kasumi-1 wasimplanted subcutaneously into athymic nude mice. Some tumor-bearing micewere treated with LOR-253 HCl at 30 mg/kg (15 mg/kg twice per day) fortwo consecutive days per week for four weeks (the “LOR-253 treatmentgroup”). LOR-253 was administered by intravenous (i.v.) bolus injectionas the hydrochloric salt form (LOR-253-HCl) formulated in 20%Polyethylene Glycol 400, 10% Propylene Glycol, and 10% Solutol HS in 60%water. Other tumor-bearing mice were treated with a formulation that didnot have LOR-253 (the “vehicle control group”).

The major endpoint was to observe for tumor growth inhibition aftertreatment with LOR-253 HCl and compare the anti-tumor effects ofdifferent dosing schedules. Tumor sizes were measured three times perweek from day 10 after the tumor cell inoculation. Tumors were measuredin three dimensions using calipers and the volume was expressed inmillimeters cubed using the formula: V=0.5a×b×c, where a, b, and c arethe length, width, and height of the tumor, respectively. Mean tumorvolumes+/−standard error (SE) were calculated from each measurement andthen plotted in a standard graph to compare the anti-tumor efficacy ofdrug treatment to that of control (FIG. 15). Toxicity was assessed byclinical observations and by measurement of mouse body weight in gramstwice per week over the course of the study. Mean body weights werecalculated from each measurement and then plotted to compare body weightchanges in the drug treatment group to that of control (FIG. 16).

The tumor inhibition results as shown in FIG. 15 demonstrate that theLOR-253 treatment group produced a statistically significantly increasedinhibitory effect in this model compared to the vehicle control group(p=0.028 by Student's t-test). In addition, the toxicity results asshown in FIG. 16 demonstrate that the mice in the treatment group didnot show body weight loss. The mice in the treatment group also did notshow other overt signs of toxicity. These results indicate that thistreatment schedule was well tolerated. LOR-253 HCl showed significanttumor growth inhibition as a single agent at twice per week dosingwithout obvious signs of toxicity, suggesting that LOR-253 HCl has asufficient therapeutic window and that this agent is a potentialchemotherapeutic agent for the treatment of AML.

Example 15 In Vivo Efficacy of LOR-253 HCL in an HL-60 AML XenograftModel

The anti-tumor activity of LOR-253 HCl, as a single agent and incombination with azacitidine, was evaluated in another in vivo animalmodel of human AML. The human AML cell line HL-60 was implantedsubcutaneously into athymic nude mice. Tumor-bearing mice were treatedwith LOR-253 HCl alone or in combination with azacitidine, withazacitidine alone, or with a negative control vehicle. The detailedtreatment conditions are as follows.

-   -   Group 1: Negative control group. LOR-253 HCl control vehicle,        2×/day, 3 cycles of 2 consecutive days, 5 days between cycles,        i.v., plus 1% D-mannitol every four days by s.c. injection    -   Group 2: Azacitidine (in 1% D-mannitol) at 10 mg/kg 1× on days        1, 4, 8, 11, 15 and 18 by subcutaneous (s.c.) injection    -   Group 3: LOR-253 HCl at 15 mg/kg bid for 3 cycles, each cycle is        2 consecutive days of dosing per week with 5 days of non-dosing,        i.v., n=9 (2T-5B)    -   Group 4: LOR-253 HCl at 15 mg/kg 2×/day (bid) for 3 cycles, each        cycle is 1 day of dosing per week with 6 days of non-dosing,        i.v., n=9 (1T-6B)    -   Group 5: Combination of LOR-253 (2T-5B) and azacitidine. LOR-253        HCl at 15 mg/kg bid for 3 cycles, each cycle is 2 consecutive        days of dosing per week with 5 days of non-dosing, i.v., n=9        (2T-5B) plus azacitidine at 10 mg/kg 1× every four days by s.c.        injection    -   Group 6: Combination of LOR-253 (1T-6B) and azacitidine. LOR-253        HCl at 15 mg/kg bid for 3 cycles, each cycle is 1 day of dosing        per week with 6 days of non-dosing, i.v., n=9 (1T-6B) plus        azacitidine at 10 mg/kg 1× every four days by s.c. injection

The major endpoint was to observe for tumor growth inhibition aftertreatment with LOR-253 HCl in combination with azacitidine. Tumor sizeswere measured three times per week. Tumors were measured in threedimensions using calipers and the volume was expressed in millimeterscubed using the formula: V=0.5a×b×c, where a, b, and c are the length,width, and height of the tumor, respectively. Mean tumorvolumes+/−standard error (SE) were calculated from each measurement andthen plotted in a standard graph to compare the anti-tumor efficacy ofdrug treatment to that of control (FIG. 17).

The tumor inhibition results as shown in FIG. 17 demonstrate thatLOR-253 HCl administered alone at 15 mg/kg twice per day for either one(Group 4) or two (Group 3) consecutive days per week inhibited growth ofHL-60 tumors to approximately the same extent as or slightly more thanazacitidine alone. Both once and twice weekly dosing of LOR-253 HCl incombination with azacitidine (Group 6 and Group 5, respectively)resulted in significantly higher levels of tumor growth inhibitioncompared to either single agent alone (p=0.0002 and p=0.0006 for 1× and2×LOR-253 HCl treatment, respectively; compared to control, asdetermined by Student's t-test). FIG. 18 and FIG. 19 show the tumor sizedata from individual animals at the beginning (Day 1) and end (Day 19)of this study.

Because LOR-253 HCl in combination with azacitidine resulted in evenhigher levels of tumor growth inhibition than either single agent alone,LOR-253 HCl may also provide additive anticancer efficacy to a standardof care chemotherapeutic for hematological malignancies.

Example 16 In Vivo Efficacy of LOR-253 HCL in a KG-1 AML Xenograft Model

The anti-tumor activity of LOR-253 HCl was evaluated in yet another invivo animal model of human AML. Xenograft model mice for AML cell lineKG-1 was generated with the same method as in Examples 14 and 15, andwere treated with LOR-253 HCl or control according to the followingregime.

Control-IV—Day 1

LOR-253—15 mg/kg-iv, bid, 2T/wk—Day 1

Control-IV—Day 8

LOR-253—15 mg/kg-iv, bid, 2T/wk—Day 8

Control-IV—Day 13

LOR-253—15 mg/kg-iv, bid, 2T/wk—Day 13

Control-IV—Day 16

LOR-253—15 mg/kg-iv, bid, 2T/wk—Day 16

Control-IV—Day 20

LOR-253—15 mg/kg-iv, bid, 2T/wk—Day 20

Control-IV—Day 26

LOR-253—15 mg/kg-iv, bid, 2T/wk—Day 26

Tumor sizes were measure as described in Examples 14 and 15, and theresults are shown in FIG. 20. LOR-253 HCl showed significant tumorgrowth inhibition as a single agent in this AML animal model as well.

Example 17 In Vivo Efficacy of LOR-253 HCL in a THP-1 AML XenograftModel

The anti-tumor activity of LOR-253 HCl, as a single agent and incombination with azacitidine, was evaluated in yet another in vivoanimal model of human AML. Xenograft model mice for AML cell line THP-1was generated with the same method as in Examples 14 and 15, and weretreated with LOR-253 HCl alone or in combination with azacitidine, withazacitidine alone, or with a negative control vehicle, and tumor sizeswere measured as described in Examples 14 and 15. The detailed treatmentconditions are shown as follows, and the results are shown in FIG. 21.

-   -   Group 1 Control: received i.v. treatments with LOR-253 control        vehicle (CV) on Days 1, 2, 8, 9, 15 &16; received subcutaneous        (SC) treatments with Azacitidine control vehicle on Days 1, 4,        8, 11, 15, 18, 22, 25, 29 &32; and received intraperitoneal (IP)        treatments with LOR-253-CV on Days 22, 23, 24, 25, 29&30.    -   Group-2—LOR-253: received i.v. treatments on Days 1, 2, 8, 9, 15        &16; received IP treatments on Days 22, 23, 24, 25, 29&30    -   Group-3—Azacitidine: received SC treatments on Days 1, 4, 8, 11,        15, 18, 22, 25, 29 &32.    -   Group-4—Combination: received i.v. treatments with LOR-253 on        Days 1, 2, 8, 9, 15 &16; received IP treatments with LOR-253 on        Days 22, 23, 24 &25; and received SC-treatment with Azacitidine        on Days 1, 4, 8, 11, 15, 18, 22 &25

The tumor inhibition results of this Example demonstrate that LOR-253HCl administered alone inhibited growth of THP-1 tumors to about thesame extent as or slightly more than azacitidine alone. When used incombination with azacitidine, LOR-253 HCl again resulted insignificantly higher levels of tumor growth inhibition.

The disclosures, including the claims, figures and/or drawings, of eachand every patent, patent application, and publication cited herein arehereby incorporated herein by reference in their entireties.

Unless defined otherwise, all technical and scientific terms herein havethe same meaning as commonly understood by one of ordinary skill in theart to which this invention belongs. Although any methods and materials,similar or equivalent to those described herein, can be used in thepractice or testing of the present invention, the preferred methods andmaterials are described herein. All publications, patents, and patentpublications cited are incorporated by reference herein in theirentirety for all purposes.

The publications discussed herein are provided solely for theirdisclosure prior to the filing date of the present application. Nothingherein is to be construed as an admission that the present invention isnot entitled to antedate such publication by virtue of prior invention.

While the invention has been described in connection with specificembodiments thereof, it will be understood that it is capable of furthermodifications and this application is intended to cover any variations,uses, or adaptations of the invention following, in general, theprinciples of the invention and including such departures from thepresent disclosure as come within known or customary practice within theart to which the invention pertains and as may be applied to theessential features hereinbefore set forth and as follows in the scope ofthe appended claims.

What is claimed:
 1. A method of treating a myelodysplastic syndrome(MDS) in a human subject, comprising administering to a human subjectidentified as suffering from a MDS an effective amount of a compound ora pharmacologically acceptable salt or solvate thereof, said compoundhaving formula I:

wherein R¹ is C1-C4 alkyl; and R² is halogen.
 2. The method of claim 1,wherein the human subject has an ineffective production of myeloid bloodcells.
 3. The method of claim 1, wherein the human subject has ananemia.
 4. The method of claim 1, wherein the human subject has lowblood counts caused by bone marrow failure.
 5. The method of claim 1,wherein R¹ is methyl, isopropyl, or t-butyl.
 6. The method of claim 5,wherein said compound has a formula selected from the group consistingof


7. The method of claim 5, wherein said compound has formula II:


8. The method of claim 1, wherein said human subject has one or moresymptoms of MDS.
 9. The method of claim 1, wherein said MDS is high-riskMDS.
 10. The method of claim 1, wherein said compound is administered aspart of a combination therapy.
 11. The method of claim 10, wherein thecombination therapy comprises radiotherapy.
 12. The method of claim 10,wherein the combination therapy comprises chemotherapy.